Single-domain antigen-binding antibody fragments derived from llama antibodies

ABSTRACT

A phage display library of variable heavy domain (V H H or VH) fragments (sdAb fragments) derived from the antibody repertoire of a non-immunized llama is disclosed. The sdAb fragments of the library are characterized by the absence of cysteine residues in complementarity determining regions (CDRs) and a very low presence of residues of glutamic acid, arginine and glycine at positions 44, 45 and 47 respectively, of the VL interface of the variable heavy domain V H H. The large size of the library (in the order of 10 9 ) makes it a source of antigen-binding fragments having high affinity to almost any antigen of interest. The library is preferably generated using a modified fd-tet phage growing in plaques in the absence of a tetracycline.

FIELD OF THE INVENTION

[0001] The invention relates to antigen-binding proteins, in particularto antigen-binding fragments of antibodies derived from a naïve libraryof llama antibodies and to a phage display library of such fragments.More particularly, the present invention relates to antigen-bindingfragments of llama antibodies comprising at least a part of the variableheavy domain (VH or V_(H)H) of antibodies derived from a naïve libraryof llama antibodies and to a phage display library of such fragments.

BACKGROUND OF THE INVENTION

[0002] The immune system in vertebrates provides a defense mechanismagainst foreign intruders, such as foreign macromolecules or infectingmicroorganisms. The foreign invaders (antigens), both macromolecules(proteins, polysaccharides, or nucleic acids) and microbes (viruses orbacteria), are recognized through specific binding of the proteins ofthe host immune system to specific sites on the antigen surface, knownas antigenic determinants.

[0003] As part of the immune system, B-cells of vertebrate organismssynthesize antigen-recognizing proteins known as antibodies orimmunoglobulins (Ig). According to the clonal selection theory, anantigen activates those B-cells of the host organism that have on theirsurface immunoglobulins that can recognize and bind the antigen. Thebinding triggers production of a clone of identical B-cells that secretesoluble antigen-binding immunoglobulins into the bloodstream. Antibodiessecreted by B-cells bind to foreign material (antigen) to serve as tagsor identifiers for such material. Antibody-tagged antigens are thenrecognized and disposed of by macrophages and other effector cells ofthe immune system or are directly lysed by a set of nonspecific serumproteins collectively called complement. In this way a small amount ofantigen can elicit an amplified and specific immune response that helpsto clear the host organism of the source of antigen. Through a complexprocess of gene splicing combined with additional mutation mechanisms,human B-cells have been estimated to produce a “library” (repertoire) ofmore than a billion (10⁹) different antibodies that differ in thecomposition of their binding sites.

[0004] For most vertebrate organisms, including humans and murinespecies, their antibodies show a common structural pattern whichconsists of two identical light polypeptide chains and two identicalheavy polypeptide chains linked together by disulfide bonds and numerousnon-covalent interactions, resulting in a Y-shaped molecule. In humans,there are two different classes (isotypes), λ and κ, of the lightchains, with no known functional distinction between them. The heavychains have five different isotypes that divide immunoglobulins intofive different functional classes (IgG, IgM, IgA, IgD, IgE), each withdifferent effector properties in the elimination of antigen.

[0005] Of the above five classes, immunoglobulins of the IgG class arethe major type in normal serum of humans and many other species and havethe four-chain structure shown schematically in FIG. 1. Each chain of anIgG molecule is divided into domains of about 110 amino acid residues,with the light chains having two such domains and the heavy chainshaving four. Comparison of amino acid sequences between different IgGsshows that the amino-terminal domain of each chain (both light andheavy) is highly variable, whereas the remaining domains havesubstantially constant sequences. In other words, the light (L) chainsof an IgG molecule are built up from one amino-terminal variable domain(VL) and one carboxy-terminal constant domain (CL), and the heavy (H)chains from one amino-terminal variable domain (VH) followed by threeconstant domains (CH1, CH2, and CH3).

[0006] The variable domains are not uniformly variable throughout theirlength. Three small regions of a variable domain, known as hypervariableregions (loops) or complementarity determining regions (CDR1, CDR2, andCDR3) show much more variability than the rest of the domain. Theseregions, which vary in size and sequence among various immunoglobulins,determine the specificity of the antigen-antibody interaction. Thespecificity of an antibody of the type shown in FIG. 1 is determined bythe sequence and size of six hypervariable loops (regions), three in theVL domain and three in the VH domain.

[0007] By partial digestion with papain, which cleaves the heavy chainsin the hinge region, the IgG molecule can be broken down into twoidentical Fab fragments (Fragment, antigen binding) and one Fc fragment(Fragment, crystallizes easily). Each Fab fragment comprises onecomplete light chain (consisting of VL and CL domains) linked by adisulfide bridge and noncovalent interactions to a fragment of the heavychain consisting of VH and CH1 domains. The Fc fragment comprises CH2and CH3 domains from both heavy chains, also linked by disulfide bridgesand noncovalent interactions. The part of the Fab fragment consisting ofvariable domains of the light and the heavy chain (VL and VH) is knownas Fv fragment (Fragment, variable). In an Fv fragment, the variabledomains VL and VH are not covalently bound. In an scFv (single chain Fv)fragment, the VL and VH domains are covalently linked by a short peptidelinker (spacer), usually 15 to 20 amino acids long, introduced at thegenetic level (see FIG. 2).

[0008] scFv fragments are recombinant fusion proteins and are producedby techniques of genetic engineering, by expressing in a suitable host,usually in bacteria, a chimeric gene coding for the fragment. Variousother recombinant antibody fragments have been designed to substitutefor large intact immunoglobulin molecules (see FIG. 2). Other than scFvfragments, these options include Fab or Fv fragments that are stabilizedor covalently linked using various strategies (see, for example, Bird etal., Science, 242, 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci.USA, 85, 5879-5883 (1988); Glockshuber et al., Biochemistry, 29,1362-1376 (1990); Jung et al., Proteins, 3547 (1994); Reiter et al.,Biochemistry, 5451-5459, 18327-18331 (1994); Young et al., FEBS Left.,135-139 (1995)). Small antigen-binding fragments of natural antibodiesare advantageous for medical applications, for example cancer targetingand imaging, when small antigen-biding molecules are required topenetrate into solid tumors.

[0009] Recent advances in gene technology have greatly facilitated thegenetic manipulation, production, identification and conjugation ofrecombinant antibody fragments and broadened the potential utility ofantibodies as diagnostic and therapeutic agents. Of particularimportance to such applications is the possibility to alter the finespecificity of the antibody binding site, to create small stableantigen-binding fragments, to prepare fusion proteins combiningantigen-binding domains with proteins having desired therapeuticproperties, for the purpose of immunotargeting, or to “humanize”antibodies of other species, for example murine antibodies (see FIG. 2).

[0010] The genetic engineering has also made possible to screen in vitrofor antibodies having a predetermined binding specificity. This may beachieved by constructing first a gene library of antibodies or antibodyfragments, for example by polymerase chain reaction (PCR)-amplificationof cDNA derived from B-lymphocytes using suitable primers, or by invitro gene synthesis. The gene library may contain sequencescorresponding to certain fragments of natural antibodies, or randomizedantigen-binding regions, or new combinations of heavy/light chains, thuscreating the potential for generating antibodies which could never beobtained from natural sources, for example, antibodies to highly toxicsubstances or antigens tolerated by the human immune system. By randomor designed mutations, the affinity or specificity of the antigenbinding can be manipulated, for example, to reach affinities neverobserved with natural antibodies.

[0011] To screen a gene library, which may contain many millions or evenbillions of different clones, for genes of antibodies having the desiredbinding specificity, a selection system comparable to that of the immunesystem is required. Such a selection system can be achieved by insertingthe library genes into the genome of microorganisms capable ofdisplaying on their surface the antibody corresponding to the insertedgene, in analogy to the expression of an immunoglobulin antigen receptoron the surface of a B-cell. Microorganisms most frequently used forproviding such a display are filamentous bacteriophages, such as fd orM13 phages (phage display). The collection of phage particles havinginserted genes of a library of proteins, such as antibodies, anddisplaying these proteins on the particles' surface is known as a phagedisplay library. The display of the library of antibodies on the surfaceof phage particles provides a physical link between the antigen-bindingfunction of an antibody and the antibody gene. Using the affinity to apreselected antigen, the whole organism (phage) displaying this affinitycan be identified and separated out of billions of non-specific clones,usually through binding to the antigen immobilized on a support,technique usually referred to as panning (see, for example, Scott etal., Science, 249, 386-390 (1990); Winter et al., Annual Rev.Immunology, 12, 433-455 (1994)). Phage clones binding to the antigen canbe then amplified and used to produce the specific antibody or antibodyfragment in E. coli or in other suitable organism.

[0012] For naturally occurring antibodies, there are examples that wholeheavy chains alone retain a significant binding ability in the absenceof light chains. It is also well established, from structural studies,that the CDR3 of the heavy variable domain generally contributes themost to antigen binding, because CDR3 amino acid residues areresponsible for most of the surface contact area and molecularinteraction with the antigen (Padlan, E. A., Mol. Immunology, 31,169-217 (1984); Chothia et al., J. Mol. Biol., 196, 904-917 (1987);Chothia et al., J. Mol. Biol., 186, 651-663 (1985)). Less bindingactivity was observed for light chain. In view of these findings,attempts were made to isolate single VH domains. For example, VH domainswere isolated from expression libraries derived from immunized mice(Ward et al., Nature, 341, 544-546 (1989)). In another report,antigen-binding VH domains were rescued from an antibody phage librarythat was made from a vaccinated patient (Cai et al., Proc Natl. Acad.Sci. USA, 93, 6280-6285 (1996)). Antigen-binding antibody fragmentsconsisting of a single VH domain, known as dAbs or sdAbs (single-domainantibodies), are becoming an attractive alternative to single chain Fv(scFv) fragments. Despite smaller binding surface, their demonstratedaffinity is comparable to that demonstrated by scFv fragments (Davies etal., Biotech., 13, 475-479 (1995)). Because of their smaller size, beinghalf of the size of scFvs, sdAbs are amenable to detailed NMR structuralstudies (Davies et al., FEBS Letters, 339, 285-290 (1994)).Additionally, due to their simpler structure, sdAbs are more stable andhave simpler folding properties.

[0013] Recently, a new class of antibodies known as heavy chainantibodies (HCA, also referred to as two-chain or two-chain heavy chainantibodies) have been reported in camelids (Hamers-Casterman et al.,Nature, 363, 446-448 (1993); see also U.S. Pat. No. 5,759,808; U.S. Pat.No. 5,800,988; U.S. Pat. No. 5,840,526; and U.S. Pat. No. 5,874,541).Compared with conventional four-chain immunoglobulins of IgG-type, whichare also produced by camelids, these antibodies lack the light chainsand CH1 domains of conventional immunoglobulins. One of the salientfeatures of these naturally occurring heavy chain antibodies is thepredominant presence of Glu, Arg and Gly at VL interface positions 44,45 and 47 (Kabat numbering), respectively, of their variable domain(designated V_(H)H). The same positions in the variable domain of theheavy chain of conventional four-chain antibodies (designated VH) arealmost exclusively occupied by Gly, Leu and Trp. These differences arethought to be responsible for the high solubility and stability ofcamelid HCA variable domain (V_(H)H), as compared with the relativeinsolubility of VH domain of the conventional four-chain antibodies. Twomore salient features of camelid V_(H)H domains are their comparativelylonger CDR3 and high incidence of cysteine pairs in CDRs. It appearsthat cysteine pairs mediate the formation of a disulfide bridge and aretherefore involved in modulating the surface topology of the antibodycombining site. In the crystal structure of a camel sdAb-lysozymecomplex, a rigid loop protruding from the sdAb and partly stabilized bya CDR disulfide linkage extends out of the combining site and penetratesdeeply into the lysozyme active site (Desmyter et al., Nature Struct.Biol., 3, 803-811 (1996)).

[0014] More recently, a number of camelid sdAbs phage display librarieshave been generated from the V_(H)H repertoire of camelids immunizedwith various antigens (Arbabi et al., FEBS Letters, 414, 521-526 (1997);Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decanniere et al.,Structure, 7, 361-370 (1999)). By creating polyclonal libraries, manyhighly soluble sdAbs with high affinity and specificity have beenisolated. However, it has been questioned whether sdAbs with desiredaffinity and defined conformations can be generated in the absence ofprior immunization, i.e., with a naïve library (Lauwereys et al.,supra). Immunization of domesticated valuable animals, such as camelids,raises serious ethical implications related to experiments with animals.Moreover, this approach has serious drawbacks because most of thepathogenic antigens cannot be injected into camelids, as this couldendanger their lives. Considering the above drawbacks and limitations ofthe prior art, there exists a strong need for the generation of phagedisplay libraries of sdAb antibody fragments derived from naïvelibraries of camelid antibodies, in particular sdAb fragments of camelidheavy chain antibodies, which libraries may become a universal source ofsdAbs for in vitro selection against any antigen of interest as atarget.

SUMMARY OF THE INVENTION

[0015] The present invention has overcome the above-discussed prior artlimitations by generating a large size (in the order of 10⁹) phagedisplay library of antibody fragments of a non-immunized llama, whichfragments comprise at least a part of the variable heavy domain (VH orV_(H)H domain) of llama antibodies. In a preferred embodiment, thefragments consist essentially of the variable heavy domain (VH or V_(H)Hof llama antibodies (sdAb fragments). This library possesses a number ofunique features which distinguish it from similar libraries generatedfrom other camelids. The large size of the library considerablyincreases the probability of isolating therefrom antigen-bindingfragments having high affinity to almost any predetermined target(antigen) of interest. This has been demonstrated by isolating from thelibrary fragments binding specifically to several preselected antigensas targets.

[0016] Thus, according to one aspect, the invention provides a phagedisplay library of antigen-binding fragments of llama antibodies, saidfragments comprising at least a part of the variable heavy domain (VH orV_(H)H) of the antibodies. Preferably, the antigen-binding fragmentsconsist of a complete variable heavy domain (VH or V_(H)H) of theantibodies (sdAb fragments)

[0017] According to another aspect, the invention provides anantigen-binding fragment of a llama antibody, said fragment comprisingat least a part of the variable heavy domain (VH or V_(H)H) of theantibody. Preferably, the antigen-binding fragment consists of acomplete variable heavy domain (VH or V_(H)H) of the antibody (sdAbfragment).

[0018] According to yet another aspect, the invention provides a cDNAlibrary comprising nucleotide sequences coding for antigen-bindingfragments of llama antibodies, said library obtained by isolatinglymphocytes from a biological sample obtained from a non-immunizedllama; isolating total RNA from the lymphocytes; reverse-transcribingand amplifying RNA sequences coding for the antigen-binding fragments;cloning the amplified cDNA in a vector; and recovering the obtainedclones. Preferably, the antigen-binding fragments consist of a completevariable domain (VH or V_(H)H) of the antibodies (sdAb fragment) and thecloning vector is a filamentous bacteriophage.

[0019] According to yet another aspect, the invention provides a processfor the preparation of an antigen-binding fragment of a llama antibody,said fragment binding to a predetermined antigen, said processcomprising the steps of isolating lymphocytes from a biological sampleobtained from a non-immunized llama; isolating total RNA from thelymphocytes; reverse-transcribing and amplifying RNA sequences codingfor antigen-binding fragments; cloning the cDNA sequences so obtainedinto a first vector, said first vector capable of a surface display ofthe corresponding antigen-binding fragments; subjecting the clones toantigen affinity selection and recovering clones having the desiredaffinity; for the recovered clones, amplifying DNA sequences coding forantigen-binding fragments; cloning the amplified DNA sequences into asecond vector; transforming prokaryotic cells with the second vectorunder conditions allowing expression of DNA coding for antigen-bindingfragments; and recovering the antibody fragments having the desiredspecificity.

[0020] Other advantages, objects and features of the present inventionwill be readily apparent to those skilled in the art from the followingdetailed description of preferred embodiments in conjunction with theaccompanying drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a schematic representation of a typical four-chainIgG-type immunoglobulin (antibody) showing (a) the structure andarrangement of heavy and light chains and the approximate positioning ofinterchain disulfide bonds, and (b) the organization of the antibodymolecule into paired domains.

[0022]FIG. 2 is a schematic representation of various modifications andfragments of IgG-type antibodies, and antigen-binding fusion proteinsderived from such fragments.

[0023]FIG. 3 is a schematic representation of steps involved inconstruction of the phage display library of llama sdAb antibodyfragments according to the present invention. For simplicity, only thecoding sequences of the mRNA transcripts are shown. A, a: heavy chainmRNA of conventional four-chain (A) and two-chain heavy chain (a)antibodies; B, b: RT-PCR product derived from A and a, respectively; c:V_(H)H derived from heavy chain antibodies. Variable (VH) and constant(CH) domains are marked with dark and light shading, respectively.

[0024]FIG. 4 is a bar graph showing fractional occurrence of the CDR3lengths. Gray bars represent data according to the present invention,whereas the white bars represent the published data for llama V_(H)H (Vuet al., Mol. Immunol., 34, 1121-1131 (1997)).

[0025]FIG. 5 is a graph showing global fitting to 1:1 interaction modelof the binding of Yst9.1 scFv to immobilized Bruc.C6 sdAb fragment at20, 100, 200, 300, 400, and 600 nM. Open circle lines representexperimental data points, whereas solid lines represent the fit.

[0026]FIG. 6 is a graph showing overlays of sensograms (A) and theScatchard plot derived therefrom (B) for the binding of TNG.p1779 sdAbfragment (2.5 (f), 7.5 (e), 10 (d), 15 (c), 20 (b) and 30 (a) μM) tocaptured biotinylated p1779 peptide.

[0027]FIG. 7 is a graph showing the Scatchard plot derived fromsensograms for the binding of TNG.PTH50 sdAb fragment to capturedbiotinylated PTH2 peptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In the following, positions of amino acid residues in antibodiesand antibody fragments are indicated according to the Kabat numbering.

[0029] The present invention provides a large size (in the order of 10⁹)phage display library of single-domain fragments of variable heavydomains (VH and V_(H)H) of llama antibodies. The library, which has beengenerated using lymphocytes of a non-immunized animal (naïve library),can be used for in vitro selection against any antigen of interest as atarget. The size of the library makes it highly probable that anantibody specific to the target will be identified among the library'ssdAb fragments. This utility of the library has been demonstrated byisolating therefrom sdAbs binding specifically to various preselectedantigens as targets.

[0030] The choice of a naïve library as the source of llama antibodieswas based in part on the fact that the immune system of camelids hasevolved over time in harsh environments and that its uniquephysiological and morphological features have helped the camelids towithstand water scarcity, adapt to climate extremes and develop anatural resistance to deadly viral diseases. The sero-epidemiologicalstudies have confirmed that camelids produce antibodies to a greatnumber of pathogenic viruses without developing the disease (Werney etal., Infectious Diseases of Camelids, Blackwell's Wissenschaft Verlag,Berlin (1995)). This means that antibodies of therapeutic importance canbe isolated from the antibody repertoire of camelids without priorimmunization with potentially dangerous pathogens or fragments thereof.

[0031] Another advantage of choosing a naïve library as the source ofllama antibodies concerns anti-idiotypic antibodies. An anti-idiotypicantibody (a second antibody) recognizes the idiotope of another antibody(a first antibody) as an antigen, meaning that the first antibodyrecognizes in turn the second (anti-idiotypic) antibody as its antigen.Anti-idiotypic antibodies have gained a widespread clinical use, e.g.,in vaccine development for cancer and cholera (Grant et al., Clin.Cancer Res., 1319-1323 (1999); Herlyn et al., Ann. Med., 66-78 (1999);Maxwell-Armstrong et al., Br. J. Surg., 149-154 (1998); Pierre et al.,Acta Gastroenterol. Belg., 430-436 (1992)) and in autoimmune diseasetherapy (Perosa et al., Clin. Exp. Rheumatol., 201-210 (1997). They havealso been shown to increase the protective immune response againstparasites, bacteria and viruses (Feodorova et al., J. Med. Microbiol.,751-756 (1999) and references therein). Since the original antigens(i.e., cancer, bacterial or viral antigens) may have been weakly- ornon-immunogenic or toxic to the cells, anti-idiotypic antibodies havebeen used in their place to provide immune protection against diseases.However, in almost all cases reported to date, anti-idiotypic antibodieshave been developed by immunization. The present invention eliminatesthe step of immunization and allows isolation of anti-idiotypicantibodies of potential diagnostic and therapeutic value from a naïvelibrary.

[0032] Among the camelids, llama is the smallest animal which cansurvive in a severe, cold climate. Lymphocytes of a llama from a farmlocated in Osgoode (Canada) have been used to generate the phage displaylibrary of variable heavy domains of llama antibodies. From thislibrary, sdAbs binding specifically to several preselected antigens havebeen subsequently isolated and characterized.

[0033] Construction of a Naïve Llama sdAb Phage Display Library

[0034]FIG. 3 depicts a schematic representation of steps involved in theconstruction of the V_(H)H-derived sdAb phage display library. As thefirst step, lymphocytes from the fresh blood of llama (from a farmlocated at Osgoode, Ontario, Canada) were prepared and their RNA wasisolated using techniques well known to those skilled in the art.RT-PCRs (reverse transcriptase-polymerase chain reactions) wereperformed using primers annealing at the 5′ end of VH or V_(H)H and CH2genes of IgG. The amplified products were separated and fragments of theexpected size derived from conventional IgG (˜900 bp) and heavy chainIgG (˜600 bp) were observed on the agarose gel. The smaller fragment wasgel purified and used in a second PCR to amplify the V_(H)H genes. Theamplification products were cloned into fd-tet (GIIID) vector, betweenthe leader signal and gene III, to produce fusion proteins, which weredisplayed on the filamentous phage particles using a modified procedure.

[0035] As is well known to those skilled in the art, the probability ofisolating a protein with high affinity or specificity against a target(antibody) of interest increases with the size of the library.Generally, two different types of vectors are used for generating phagedisplay libraries: phagemid vectors and phage vectors. Libraries havingsize in the order of 10⁸ can be constructed with relative ease usingphagemid vectors. However, a phagemid-based libraries suffers from someserious drawbacks. First, phagemid vectors provide typically amonovalent display and therefore may not select for lower binding (oflower affinity), but potentially important antibody fragments. Second, aphagemid-based library allows for the enrichment of phage particlesdisplaying deleted versions of the antibody fragments. Such particles,often with no binding activity, are preferably selected during thepanning process over those displaying the full-length fragments andtherefore obscure the process of selection of the full-length binders.Third, constructing a phagemid-based library requires a helper phage andtherefore library construction, panning and downstream phage bindingassays become a far more complicated and tedious task. For these reasonsthe use a phage vector for the library construction is preferred.

[0036] One of the most widely used phage vectors is fd-tet (Zacher IIIet al., Gene, 9, 127-140 (1980)) which consists of fd-phage genome, plusa segment of Tn10 inserted near the phage genome origin of replication.Tn10 contains a tetracycline resistance gene, tetA, and thus conferstetracycline resistance to the host cells carrying the fd-tet vector. Ithas often been observed that the size of the fd-tet based library wasgenerally low (in the range of 10⁵-10⁶) (Harrison et al., Methods inEnzymology [Ed. Abelson, J. N.], 267, 83-109 (1996); Krebber et al.,FEBS Letters, 377, 277-331 (1995)), possibly due to the toxic effect ofteta gene product on the host cells. According to the modified procedureof the present invention, the library was propagated as plaques in theabsence of tetracycline, resulting in a llama V_(H)H library of size ofapproximately 8.8×10⁸. This is the largest size library ever obtainedusing fd-tet vector. Due to its size, the library has an enhancedprobability of selecting therefrom proteins (antibody fragments) bindingto almost any given target (antigen).

[0037] It would be known to those skilled in the art that, at least inprinciple, the display library of the invention could be generated usingvectors other than phages, such as bacteria (e.g., E coli) (Daugherty etal., Protein Eng., 613-621 (1999); Georgiou et al., Nat. Biotechnol.,29-34 (1997)) or yeast (e.g., Saccharomyces cerevisiae) (Kieke et al.,Proc. Natl. Acad. Sci. USA., 5651-5656 (1999); Kieke et al., ProteinEng., 1303-1310 (1997); Cho et al., J. Immunol. Methods, 179-188 (1998);Boder et al., Nat. Biotechnol., 553-557 (1997)). Obtaining largelibraries, comparable in size to phage display libraries, is, at leastin theory, possible using these vectors. However, these display systemshave not been of a widespread use, as they require expensive flowcytometry cell sorting instruments for selection. In addition, the E.coli display system is not suitable for panning against largemacromolecules, such as proteins, due to the interference of thelipopolysaccharide layer of E. coli with the binding process (Boder etal., supra). Surface display of an scFv on mammalian cells has also beenreported (Rode et al., J. Immunol. Methods, 151-160 (1999); Rode et al.,BioTechniques, 650, 652-656, 658 (1996)). However, no antibody libraryhas been so far constructed using vectors other than phages, as theconstruction and screening in these alternative display systems are notas rapid or versatile as for phage display libraries.

[0038] Sequence Analysis

[0039] Colony PCR of 80 randomly selected clones showed that more than60% had the full-length V_(H)H genes (sdAbs). The identity of the VLinterface amino acids at position 44, 45 and 47 as well as the CDRssequence of 28 randomly selected sdAbs have been determined and aresummarized in Table 1. FIG. 4 shows the fractional occurrence of theCDR3 length. For comparison, previously published sequence data obtainedfrom llama HCAs are also included. Similar to the previous results, themajority of the CDRs of the sequenced sdAbs are 13-17 amino acid long,demonstrating that the llama sdAb library of the invention is derivedfrom heavy chain antibodies. However, the present library is distinct inseveral aspects from the known V_(H)H libraries. TABLE 1 CDR/H1sequences of 28 randomly selected dAbs from the llama library. The VLinterface residues at positions 37, 44, 45 and 47 are also included.Position 35 is in each case the last residue in CDR/H1 sequence. adAb 3744 45 47 CDR1/H1 CDR2 CDR3 C1 V G L W GFTFSSYYMS SEQ ID NO: 1GIYSDSSITAYADSVKG SEQ ID NO: 29 MVMGPAATGYEY SEQ ID NO: 57 C2 F E R FGRTFSNYHMG SEQ ID NO: 2 SIKWSGGNTYYADSVKG SEQ ID NO: 30GSKYGGSWSRSQDAYNY SEQ ID NO: 58 C4 F E R F GRIFSNAAMG SEQ ID NO: 3AIRWSDGNTYYADSVKG SEQ ID NO: 31 GIGTFGSSWTRADRYRY SEQ ID NO: 59 C5 Y Q RL RSIFSINTLG SEQ ID NO: 4 WITSGGATYYADSMKG SEQ ID NO: 32 RVPLDY SEQ IDNO: 60 C7 F E K F GRSFSTYRVG SEQ ID NO: 5 GINWNGVKTRYSDSMND SEQ ID NO:33 DQRFDGDDWSPSAFTR SEQ ID NO: 61 C8 F E R F GNTISGYATG SEQ ID NO: 6AVTWSGYSVYYAKSPKG SEQ ID NO: 34 VFVRTAGVPTLGEYDY SEQ ID NO: 62 C9 F G RF GGSFSNYNMG SEQ ID NO: 7 GIGWSGGRIIVADSVKG SEQ ID NO: 35TKQFFPLSN?SVWYDY SEQ ID NO: 63 C12 W K R F GRIPRNYPIG SEQ ID NO: 8GISWrSGTTYFADSVKG SEQ ID NO: 36 SERDFYTRNYYFTFESLYDY SEQ ID NO: 64 C15 FA R F GESIASFNLG SEQ ID NO: 9 AVSRTGETTDYADAVKG SEQ ID NO: 31DYNLGTFVTRKDSMYDF SEQ ID NO: 65 C16 F E R F GRTFSSVSMG SEQ ID NO: 10AINWRGVSTYYADSVKG SEQ ID NO: 38 RRNFFGNNSAGQYAY SEQ ID NO: 66 C17 L E RF GLTFGDYAMG SEQ ID NO: 11 TISRIGSTTYYADSVKG SEQ ID NO: 39 SRYVLKYDKDAYSEQ ID NO: 67 C22 F E R F GRTFSSVTMG SEQ ID NO: 12 AMTRNSGSTYYADSVKG SEQID NO: 40 KASXYGSTLYPPTGYNY SEQ ID NO: 68 C24 F E R F GRTFSRFAMG SEQ IDNO: 13 AISWSGGTTYGADSAKG SEQ ID NO: 41 GRAVSDYDY SEQ ID NO: 69 C25 Y E RL GSIFSESAMG SEQ ID NO: 14 AITLDGRTNYAYYAEG SEQ ID NO: 42 LRSRAVMDTIPNYSEQ ID NO: 70 C26 F E R F GRTFSSDAMG SEQ ID NO: 15 AISWSGGSTYYADSVKG SEQID NO: 43 DRRRYYSGSYPPSEYDY SEQ ID NO: 71 C29 V G L W GFTFSNFWMG SEQ IDNO: 16 QINTGGDITTYSDSVKG SEQ ID NO: 44 ARSVPLSDPRTYSS SEQ ID NO: 72 C30L E R V GRSFNHYIMG SEQ ID NO: 17 SIDWNSGRTNYADSVKG SEQ ID NO: 45AAAASTLVGGSYDY SEQ ID NO: 73 C31 Y E R F GLPFSTYSMG SEQ ID NO: 18VIGGGGNTYHAADSLKD SEQ ID NO: 46 DRDFTIVAGFIRSQYSPRAVEY SEQ ID NO: 74 C33F E R F GRTFSTYTMG SEQ ID NO: 19 AISRNSVGTYYRDSVKG SEQ ID NO: 47DPMYGRSVMSTRYNY SEQ ID NO: 75 C34 F D R F GYTFSSHNIG SEQ ID NO: 20AISASGGNQYYKYFAKG SEQ ID NO: 48 ATKQFSNAYSDYVHDYDY SEQ ID NO: 76 C35 F ER G GFRFAEYAIG SEQ ID NO: 21 YISTSDKTTYYSDFAEG SEQ ID NO: 49GLYYSDYKTPEYTEYVH SEQ ID NO: 77 C40 F E R F GRTFSRFAMG SEQ ID NO: 22AISWSGGTAYGADSAKG SEQ ID NO: 50 GRAVSDYDY SEQ ID NO: 78 C43 V G L WGFTFVDYSMT SEQ ID NO: 23 AINWNGRLTYYAESMKG SEQ ID NO: 51 GELYGMGSKHDYSEQ ID NO: 79 C44 V G L W GFTFSNYYMY SEQ ID NO: 24 MVNTGGGGTRYADSVRG SEQID NO: 52 DRPQSGWSNDY SEQ ID NO: 80 C45 F E R F GLTFSSYVMG SEQ ID NO: 25AIITSGRSTYYADSVKG SEQ ID NO: 53 TKWVVKRPADYNY SEQ ID NO: 61 C46 F E R FGGTFTDYAMG SEQ ID NO: 26 AINWGGYSTYYSDAVKG SEQ ID NO: 54 DPQLITTPEYNYSEQ ID NO: 82 C48 V G L W GFTFSNYYMY SEQ ID NO: 27 MVNTGGGGTRYADSVKG SEQID NO: 55 DRPQSGWSMDY SEQ ID NO: 83 C49 F E R F GNTISDYATG SEQ ID NO: 28SIGRRTGWQVYSDSVKG SEQ ID NO: 56 SQDSGFDTPVTESHLYGY SEQ ID NO: 84

[0040] Previously generated camelid sdAb libraries were characterized bytypical presence of Glu, Arg and Gly in positions 44, 45 and 47,respectively, of the VL interface of V_(H)H domain. The occurrence ofcysteine at position 45 was also frequent in V_(H)H, as opposed to VHdomain of four-chain IgGs. The present library, as shown by sequenceanalysis (Table 1), lacks these characteristics, as only one sdAb (C35)has Glu44, Arg45 and Gly47. The majority of sdAbs of the present libraryhave Arg in position 45 of the VL interface. This occurrence of Arg45 isnot unique to camelid V_(H)H, as a number of conventional antibodies,such as H1-I6 (VH) and V13 (VH), have been found to have Arg in position45 (Blier et al., J. Immunol., 139, 3996-4006 (1987); Crews et al.,Cell, 29, 59-66 (1981)). The presence of Gly at position 35 was observedto always accompany Phe at position 37, unlike a previously reportedllama library in which this pairing was observed in only 50% of thesequences. This is noteworthy in view of the fact that Gly at position35 results in local conformational changes that allow Trp101 to stackwith Arg45 in addition to engaging in aromatic-aromatic interactionsinvolving Phe37 and Trp103. For the present library, 12 of 27 sdAbs haveTrp at position 52a, whereas only 1 of the 51 previously publishedsequences have Trp at this position.

[0041] Another major difference between the present library and thepreviously reported V_(H)H libraries of camelids concerns the CDRcysteins. Previously generated libraries were characterized by a highincidence of cysteine pairs in CDRs, whereas none of the 28 sdAbs(Table 1) of the present library had any cysteine in their CDRs. Thelibrary of the invention is therefore characterized by a very lowpresence or by the absence of cysteine residues in CDRs.

[0042] Finally, the present library, which was designed and constructedto contain only antibody fragments consisting of variable heavy chaindomains (V_(H)Hs), also contains a substantial number of typicalconventional variable heavy domains (VHs) (for example, sdAbs C1, C29,C43, C44 and C48 of Table 1, some sdAbs of Table 2). This contaminationis most likely the results of PCR crossovers between the VHs and V_(H)Hsduring the step of RT-PCR (FIG. 3) (Tomlinson et al., J. Mol. Biol.,227, 776-798 (1992); Muyldermans et al., Protein Eng., 7,1129-1135(1994)). These VHs are genuine antigen binding fragments, asshown in Table 2, produced in high yield in Escherchia coli. They arehighly soluble, have excellent temperature stability profiles and do notdisplay any aggregation tendencies (Tanha et al., manuscript inpreparation; Vranken et al., submitted). The very close similarity ofthese molecules to human VHs makes them potentially very useful astherapeutic sdAbs.

[0043] For the library of the invention, amino acids of the VL interfaceare most frequently:

[0044] at position 44—Gly, Glu, Gin, Lys, Ala and Asp,

[0045] at position 45—Leu, Phe, Pro and Arg, and

[0046] at position 47—Trp, Tyr, Phe, Leu, lie, Val and Gly.

[0047] For the library of the invention, CDRs can be selected from thefollowing sequences: CDR1/H1: GFTFSSYAMS (SEQ ID NO: 85) GFTFSSYYMS (SEQID NO: 86) GFTFDEHAIG (SEQ ID NO: 87) GFTVSSNHMT (SEQ ID NO: 88)GFTFSSYHMA (SEQ ID NO: 89) GFTFSRHQMS (SEQ ID NO: 91) GFTFRTYYMN (SEQ IDNO: 92) GFIFSSYAMS (SEQ ID NO: 93) GFTFSTYAMT (SEQ ID NO: 95) GFTFSGYAMS(SEQ ID NO: 99) GFAFSNYRMT (SEQ ID NO: 100) GFTFSRYAMS (SEQ ID NO: 101)CDR2: GIEGGGGITRYADSVKG (SEQ ID NO: 102) TIKPGGGSTYYADSVKG (SEQ ID NO:103) TIDIGGGRTYADSVKG (SEQ ID NO: 104) RISSDGRNTYYADSVKG (SEQ ID NO:105) TINPGDGSTYYADSVKG (SEQ ID NO: 106) HIDTGGSTWYAASVKG (SEQ ID NO:107) TINIDGSSTYYADSVRG (SEQ ID NO: 109) GINSFGGSKYYADSVKG (SEQ ID NO:110) TINTSGRGTYYADSVKG (SEQ ID NO: 112) AINSGGGSTSYADSVKG (SEQ ID NO:113) HIDTGGGSTWYAASVKG (SEQ ID NO: 114) DINSGGDSTRNADSVKG (SEQ ID NO:115) SINSGGGSTYYADSVKG (SEQ ID NO: 116) RINSIGDRISYADSVKG (SEQ ID NO:117) CDR3: AHGGYGAFGS (SEQ ID NO: 119) YSGGALDA (SEQ ID NO: 122)LSQGAMDY (SEQ ID NO: 124) IDRERAFTS (SEQ ID NO: 127) IDWERAFTS (SEQ IDNO: 128) QGYAGSYDY (SEQ ID NO: 129) LGVPGTFDY (SEQ ID NO: 130) TNRGIFDY(SEQ ID NO: 131) TPGSSGVYEY (SEQ ID NO: 132) TQTGSHDY (SEQ ID NO: 133)QVGTAYDY (SEQ ID NO: 134) RRGSSGVYEY (SEQ ID NO: 135)

[0048] Selection Against Antibody Antigens

[0049] Special cases of antibody-antigen reactions are those in whichthe antigen (Ag) is itself an antibody (Ab), as discussed above. Singledomain anti-idioptypic (anti-id) antibody fragments have been isolatedfrom the library of the present invention using phage display technologyand an antibody serving as antigen. Such anti-Id antibody fragments havegreat potential in both evoking the immune system responses topathological antigens and in vaccine development.

[0050] Single Chain Fv-Yst9.1 (Anti-Brucella Antibody)

[0051] The above-described naïve llama phage display library was pannedagainst Yst9.1 scFv immobilized on micro-titer plates. A very highenrichment was observed in the case of anti-Brucella carbohydrate(Yst9.1 scFv), as all the 60 selected clones showed strong binding inphage ELISA to Yst9.1 scFv but no binding to the BSA control. Sequencingrevealed 17 different sdAbs, some of which, were related to each other(Table 2). For example, Bruc.B3, Bruc.B10 and Bruc.C7.3 have the sameCDR3. As another example, Bruc.C7.2, Bruc.D10 and Bruc.E6 have the sameCDR3 in addition to the first two, which share the same CDR2. Thesecommon sequences were encoded by identical nucleotides raising thepossibility that divergent sdAbs may have arisen as a result of PCRcross-over in vitro. Interestingly, the interface amino acids aregenerally Gly44, Leu45 and Trp47, typical of human/murine VH domain. Inaddition, none of the isolated sdAbs have any cysteine in CDR1, 2, or 3.

[0052] Table 2 also shows the identity of amino acids at positions 37,44, 45 and 47 of the VL interface of V_(H)H domain. Interestingly, allsdAbs shown in the table have VL TABLE 2 CDR/H1 sequences of dAbs whichwere isolated by panning the llama library against Yst9.1 scFv. TheV_(L) interface residues at positions 37, 44, 45 and 47 are alsoincluded. sdAb 37 44 45 47 CDR1/H1 CDR2 CDR3 Bruc.B3 V G L W GFTFSSYAMSSEQ ID No: 85 GIEGGGGITRYADSVKG SEQ ID NO: 102 AHGGYGAFGS SEQ ID NO: 119Bruc.B10 V G L W GFTFSSYYMS SEQ ID No: 86 TIKPGGGSTYYADSVKG SEQ ID NO:103 AHGGYGAFGS SEQ ID NO: 120 Bruc.C7.3 F G F S GFTFDLHAIG SEQ ID No: 87TIDIGGGRTYADSVKG SEQ ID NO: 104 AHGGYGAFGS SEQ ID NO: 121 Bruc.B8 V G LW GFTVSSNNNT SEQ ID No: 88 RISSDGRNTYYADSVKG SEQ ID NO: 105 YSGGALDA SEQID NO: 122 Bruc.D4.4 V G L W GFTFSSYHMA SEQ ID No: 89 TINPGDGSTYYADSVKGSEQ ID NO: 106 YSGGALDA SEQ ID NO: 123 Bruc.C7.2 F G L Y GFTFDEHAIG SEQID No: 90 HIDTGGSTWYAASVKG SEQ ID NO: 107 LSQGAMDY SEQ ID NO: 124Bruc.D10 V G L Y GFTFSRHQMS SEQ ID No: 91 HIDTGGSTWYAASVKG SEQ ID NO:108 LSQGAMDY SEQ ID NO: 125 Bruc.E6 V G L W GETFRTYYMN SEQ ID No: 92TINIDGSSTYYADSVRG SEQ ID NO: 109 LSQGAMDY SEQ ID NO: 126 Bruc.E3.1 V G LW GFIFSSYAMS SEQ ID No: 93 GINSFGGSKYYADSVKG SEQ ID NO: 110 IDRERAFTSSEQ ID NO: 127 Bruc.E7.3 V G F W GFIFSSYAMS SEQ ID No: 94GINSFGGSKYYADSVKG SEQ ID NO: 111 IDWERAFTS SEQ ID NO: 128 Bruc.C6 V G LW GFTFSTYAMT SEQ ID No: 95 TINTSGRGTYYADSVKG SEQ ID NO: 112 QGYAGSYDYSEQ ID NO: 129 Bruc.C5 V G F W GFTFSSYAMS SEQ ID No: 96AINSGGGSTSYADSVKG SEQ ID NO: 113 LGVPGTFDY SEQ ID NO: 130 Bruc.B7.1 V GL Y GFTFSRHQMS SEQ ID No: 97 HIDTGGGSTWYAASVKG SEQ ID NO: 114 TNRGIFDYSEQ ID NO: 131 Bruc.B7.1A V G P W GFTFSRYAMS SEQ ID No: 98DINSGGDSTRNADSVKG SEQ ID NO: 115 TPGSSGVYEY SEQ ID NO: 132 Bruc.D6 V G LW GFTFSGYAMS SEQ ID No: 99 SINSGGGSTYYADSVKG SEQ ID NO: 116 TQTGSRDY SEQID NO: 133 Bruc.D5 L G F W GFAFSNYRMT SEQ ID No:100 RINSIGDRISYADSVKGSEQ ID NO: 117 QVGTAYDY SEQ ID NO: 134 Bruc.F7 V G P W GFTFSRYAMS SEQ IDNo:101 DINSGGDSTRNADSVKG SEQ ID NO: 118 RRGSSGVYEY SEQ ID NO: 135

[0053] interface residues which are typical of murine or human VHs. Forexample, half of the sdAbs have Val37, Gly44, Leu45 and Trp47, which arehighly conserved in murine and human VH. In addition, all sdAbs haveVal37 and Gly44, and majority has Leu45 and Trp47. Six, three and onesdAbs are characterized by the presence of Phe45 or Pro45, Tyr45 andSer45, respectively. It is interesting to note that the presence of thesame VL interface residues in the conventional antibodies would renderthe isolated VH highly hydrophobic, resulting in their aggregation,which is not observed for llama antibodies.

[0054] With the presence of “human residues” at positions 37, 44, 45 and47, the entire sequences of the Yst9.1-specific sdAbs are veryhomologous to human VH3 family sequences. A comparison of a consensusVH3 family sequence and the Yst9.1-specific sdAbs reveals amino aciddifferences at only five positions (Table 3). One of the fivedifferences, the position 83 difference (Lys in the Yst9.1-specificsdAbs and Arg in the human consensus sequence) is conservative.Spatially, residues 6 and 108 are close and are located in the first andlast (ninth) β-strands, respectively. The other three residues arepositioned in non-CDR loops. Incorporation of some of these residuesinto an otherwise insoluble human V_(H) has rendered the domain soluble(unpublished results). TABLE 3 Amino acid differences between a humanVH3 family concensus sequence and the Yst9.1 binders listed in Table 2.Amino Acid positions are indicated in Kabat Numbers. Amino acid position6 74 83 84 108 V_(H)H Ala Ala Lys Pro Gln Human VH3 family Glu Ser ArgAla Leu

[0055] Binding Studies

[0056] One of the anti-Yst 9.1 scFv sdAbs, Bruc.C6, was shown to bespecific for its antigen by BIACORE analysis, as it bound to Yst 9.1scFv (FIG. 5). The kinetic rate constants, k_(a) and k_(d), obtained bythe global fitting of the binding data, are shown in Table 5. Thecalculated K_(d) in this case is 380 nM (Table 5). TABLE 5 Kinetic andequilibrium constants for the binding of Bruc.C6 to Yst9.1 ScFv and ofTNG.P1779 to biotinylated peptide p1779. The values were determined fromthe retrospective sensograms and Scatchard plots in FIGS. 5, 6 and 7. ND= not determined. K_(d) (M) K_(d) (M) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹)(k_(d)/k_(a)) (Scatchard plot) Bruc.C6 1.4 × 10⁴ 5.5 × 10⁻³ 3.8 × 10⁻⁷ND TNG.P1779 ND ND ND 1.1 × 10⁻⁵

[0057] Selection Against Peptide Antigens

[0058] These selection studies were carried out against peptides derivedfrom granulin A and the parathyroid hormone (PTH).

[0059] A. Granulin A-Derived Peptides

[0060] Proteins of granulin/epithelin family are thought to play a rolein inflammation, wound repair, tissue modeling and regulating enzymeactivity (Vranken et al., J. Pept. Res., 590-597 (1999); Hrabal et al.,Nat. Struct Biol., -752 (1996)). They are implicated as potentialco-factors for HIV Tat protein and in modulating the growth of humanepidermal carcinoma cells, and inhibition of their expression is knownto inhibit the tumorigenecity of certain cells. The granulin motif hasbeen found throughout the animal kingdom, in fish and insects, andencoded in the genome of a nematode worm. The motif consists of aparallel stacks of beta-hairpins pinned together by disulfide bonds. Thestructural sub-domain of granulin containing the first two beta-hairpinand spanning the first N-terminal 30 amino acids is also shared bygrowth factor proteins such as epidermal growth factors, transforminggrowth factor (TGF)-alpha, as well as the epithelial cell-specific TGF(TGF-e) which modulates the growth of human epidermal carcinoma cells.These growth factors interact with their receptors through theirN-terminal beta-hairpin sub-domain and it is believed thatepithelin/granulin family of proteins exert their growth modulatingeffect through the same subdomain, by interacting with similarreceptors. There have been continuous efforts in engineering stablesub-domains as possible drug candidates, with the aim of targetingspecific proteins in vivo. The methodology has involved a rational aminoacid substitution followed by assessing the effect of substitution onthe stability of the sub-domain by NMR studies.

[0061] Solution structure of a 30-residue N-terminal sub-domain derivedfrom carp granulin-1 has shown that the fragment forms two beta-hairpinssimilar to the one in the native protein. Unlike the carp granulin-1sub-domain, the human counterpart (Tolkatchev et al., Biochemistry,2878-2886 (2000); see also peptide p1779 in Table 6) was not stableoutside the context of the native protein and a Q20P substitution(p1781) only slightly improved its stability. A substituted versionincorporating D1V, K3H, S9I and Q20P, however, showed a well-foldedstack of two beta-hairpins as in the carp granulin-1.

[0062] As an alternative and complement to NMR studies, antibodies canbe used to probe the structural changes caused by amino acidsubstitution. The changes in the stability of a sub-domains broughtabout by amino acid substitutions may be manifested as changes in itsaffinity for an antibody probe compared to the wild type. Using peptidesp1779, p1780 and p1781 shown in Table 6 as a model system it wasdemonstrated that a sdAb isolated from the llama sdAbs phage displaylibrary by panning against p1779 may serve as a structural probe. ThesdAb binds to the p1779 peptide with a K_(d) of 10 μM, but shows nobinding to the substituted versions of the peptide (peptides p1780 andp1781), which are known to have structures different from p1779. Otherthan serving as structural probes, such sdAbs can be used, for example,to interfere with granulin binding in pathways leading to cancer cellgrowth or HIV progression. TABLE 6 Sequences of the humangranulinA-derived peptide p1779 and its substituted versions p1780 andp1781. For panning experiments the peptides were labelled at theN-terminal through a (Gly)₄ linker. Peptide Sequence p1779DVKCDMEVSCPDGYTCSRLQSGAWGCSPFT SEQ ID No: 202 p1780VVHCDMEVICPDGYTCSRLPSGAWGCSPFT SEQ ID No: 203 P1781DVKCDMEVSCPDGYTCSRLPSGAWGCSPFT SEQ ID No: 204

[0063] TABLE 4 CDR/H1 sequences of dAbs which were isolated by panningthe llama library against granuline A-derived peptides p1779 and p1781(A) and PTH peptides (B). The VL interface residues at positions 44, 45and 47 are also included. sdAb 44 45 47 CDR1/H1 CDR2 CDR3 (A) TNG.P1779Q R L GSRRSFNVMG SEQ ID No: 136 TITVGDTTSYAEAVKG SEQ ID No: 158EEWLGVRQNNY SEQ ID No: 180 TNG.P1781-1 E R L GDTFSINAYG SEQ ID No: 137AISGRGTNTFVADSVKG SEQ ID No: 159 GEY SEQ ID No: 181 TNG.P1761-2 G L WGFTFRDYWMY SEQ ID No: 138 SIYSDGSRTAYAASVKG SEQ ID No: 160 MLLGPGAPGYDYSEQ ID No: 182 TNG.P1781-3 Q R L GITFSEKHMA SEQ ID No: 139VITRGGTTNYGDSVKG SEQ ID No: 161 DFYGLGFDY SEQ ID No: 183 TNG.P1781-4 E RF ERTFNSYAAA SEQ ID No: 140 GITKNGVTYYAPSVTG SEQ ID No: 162APKYEGVSDTSSDYNY SEQ ID No: 184 (B) TNG.PTH1 E R F GRTFSSYGMG SEQ ID No:141 AMRESGADTHYADFVRG SEQ ID No: 163 LDITTAASY SEQ ID No: 185 TNG.PTH2 ER F GRTFSSYGMG SEQ ID No: 142 PMRESGADTHYADFVRG SEQ ID No: 164 TINGAARSEQ ID No: 156 TNG.PTH4 K R L GTSSGINAMV SEQ ID No: 143 TITNSGKTDYAASAKGSEQ ID No: 165 TINGAAR SEQ ID No: 187 TNG.PTH5 E R F GRTFSSYSMA SEQ IDNo: 144 AINWRSSVTAYADSVKG SEQ ID No: 166 EALPGTYGLDY SEQ ID No: 188TNG.PTH7 Q R L VSTFSIGAIG SEQ ID No: 145 GISGGGSTYYTDSVKG SEQ ID No: 167ILAGGLLAF SEQ ID No: 189 TNG.PTH8 Q R L GSTFSGNDIG SEQ ID No: 146VISDGGYTSYATSVKG SEQ ID No: 168 GGSSGTF SEQ ID No: 190 TNG.PTH9 E R FGRTFSSYGMG SEQ ID No: 147 AISWGAGTPYYADSVKG SEQ ID No: 169 TINGAAR SEQID No: 191 TNG.PTH10 E R I GRTFSDIAMA SEQ ID No: 148 AIDWNGGTTYYTTFVKGSEQ ID No: 170 LDITTAASY SEQ ID No: 192 TNG.PTH11 E R F GQTLNTYVMG SEQID No: 149 AINWRDTSTYYQDSVKG SEQ ID No: 171 TINGAAR SEQ ID No: 193TNG.PTH12 E R F GPTSITYGMA SEQ ID No: 150 AVTPSGGAAAYADSVKG SEQ ID No:172 GTELAPKTATGA SEQ ID No: 194 TNG.PTH14 E R F GGDVSTYANV SEQ ID No:151 LLSRSGRTTNYADSVKG SEQ ID No: 173 GSN SEQ ID No: 195 TNG.PTH15 Q R LGRTFGSYTNG SEQ ID No: 152 RINSAGRTMYADSVKG SEQ ID No: 174 GTVLSVATGPYGYSEQ ID No: 196 TNG.PTH18 E R F GRTFSSYGMG SEQ ID No: 153SINWRGSSTYYADSVKG SEQ ID No: 175 WGAGEDEDY SEQ ID No: 197 TNG.PTH22 Q RL GSLSRITVNG SEQ ID No: 154 IITSSGGTDYADSVKG SEQ ID No: 176 KSRDSAGLSWDYSEQ ID No: 108 TNG.PTH23 Q R V GSISSFDAMA SEQ ID No: 155IITSGGATNYADSVKG SEQ ID No: 177 LVASTVTSSVS SEQ ID No: 199 TNG.PTH50 E RF GRPFSSFAMG SEQ ID No: 156 AISASGGETYYTGSLKG SEQ ID No: 178 TINGAAR SEQID No: 200 TNG.PTH61 E R F GRTFSSYHMG SEQ ID No: 157 AINWSGDTTYYEASVKGSEQ ID No: 179 QTRPRPYGTSRAEGDYGY SEQ ID No: 201

[0064] Human Granulin A-Derived Peptides

[0065] Solution panning was performed against human granulin A-derivedpeptide, p1779, and its substituted versions, p1780 and p1781 (Table 6).After four rounds of panning against p1779, phage sdAbs from all 48clones tested were shown to bind to the target antigen. In the case ofp1781, only eight binders (four different sequences, Table 4) wereidentified. No binder was identified for p1780 even after fifth roundand performing the panning experiment two more times under differentconditions.

[0066] Sequencing of twenty-one p1779-specific sdAb genes identified onefragment, namely, TNG.P1779, which was further expressed for detailedbinding studies by BIACORE. In agreement with the phage ELISA results,TNG.P1779 was shown to be active by BIACORE analysis in whichbiotinylated p1779 was captured on a SA-coated CM5 sensor chip (FIG. 6,part A). No binding was detected to the reference surfaces on which asimilar amount of p1780 or p1781 had been captured (data not shown). AScatchard plot of the binding data gave a K_(d) of 1.1×10⁻5 M (Table 5).These results demonstrate that the TNG.P1779 behaves like a structuralprobe, sensing the structural changes, which occur in p1780 or p1781 asa result of amino acid substitutions.

[0067] B. Parathyroid Hormone-Derived Peptide

[0068] Parathyroid hormone (PTH) is the major regulator of serum calciumlevels and its use for the treatment of bone loss due to osteoporosishas been postulated. Osteoporosis, which is characterized by bone loss,strikes at any age, affects both men and women, although women withhigher frequency, and can results in hospitalization, disability anddeath (Morley et al., Current Medicinal Chemistry, 6, 1095-1106 (1999);Whitfield et al., Drugs & Aging, 15(2), 117-129 (1999)). Most of theavailable drugs slow down or stop further bone loss, but have no bonegrowth-stimulating effects, hence are not capable of replacing the lostbones.

[0069] The bone-building action of the parathyroid hormone (PTH) and itsimplications for the treatment of osteoporosis has been recentlyreviewed (Whitfield et al., supra). PTH is expressed as a 115 amino acidprecursor and secreted as a 84-residue peptide, but its bonegrowth-stimulating effects have been related to its N-terminal34-residues peptide and shown to be the case in human trials. Morerecently, mutated and cyclized PTH peptide analogues have been shown tobe more potent bone growth stimulators in in vitro studies (Morley etal., Expert Opin. Therap. Pat., 8, 30-37 (1998)). These analogues, whichhave been patented, show great promise as drugs for the treatment ofosteoporosis and are currently at the clinical trial stage. However, tomeet the regulatory requirements, the pharmacokinetics of these drugsneeds to be monitored following their administration to human subjects.This can be achieved by obtaining reagents, such as antibodies, capableof specifically recognizing the PTH analogues present in biologicalsamples. Within the past two years, attempts were made to raise suchantibodies by conventional hybridoma technology, but no success wasreported. In the present study, a number of sdAbs specific for the PTHpeptide analogues PTH1 and PTH2 (Table 7) have been isolated from thephage display library of sdAb fragments of heavy chain antibodiesderived from a naïve library of llama antibodies. TABLE 7 Sequences ofPTH1 and PTH2 peptides corresponding to N-terminal residues 17-31 (PTH1)and 1-31 (PTH2) of the human PTH. Compared to the human PTH, theseanalogs have a substitution at posi- tions 37(K37L) and a β-lactam bondconnecting the side chains of ²²E and ²⁶K. Peptide Sequence PTH1¹⁷SMERVEWLRKLLQDV³¹ SEQ ID No: 205 PTH2¹SVSEIQLMHNLGKHLNSMERVEWLRKLLQDV³¹ SEQ ID No: 206

[0070] Human PTH-Derived Peptides

[0071] Panning against PTH1 resulted in the identification of thirteendifferent sdAbs, all of which bound to PTH1 in a phage ELISA (Table 4,TNG.PTH1 through TNG.PTH18). Four binders were identified for PTH2(Table 4, TNG.PTH22, TNG.PTH23, TNG.PTH50 and TNG.PTH61). The bindingsdAbs were expressed and purified in large quantities. The expressionlevel was high and for one particular sdAb it exceeded 200 mg of proteinper liter of bacterial culture. Three sdAbs were characterized in moredetails by surface plasmon resonance and shown to bind to their targetantigens (Table 8). FIG. 7 shows the binding profile for TNG.PTH50 whichwas isolated by panning against PTH2. The calculated K_(d) for TNG.PTH50is 4.3×10⁻⁶ which is shown in Table 8. TABLE 8 Equilibrium constants forthe binding of TNG.PTH22, TNG.PTH23 and TNG.PTH50 to biotinylated PTH2.The values were determined from the respective sensograms and Scatchardplots, as shown in FIG. 7 for TNG.PTH50. sdAb K_(d) (M) TNG.PTH22 1.4 ×10⁻⁵ TNG.PTH23 5.7 × 10⁻⁵ TNG.PTH50 4.3 × 10⁻⁶

[0072] Experimental

[0073] All reagents were chemical grade purchased from variouscompanies. Unless stated otherwise, the media were prepared as described(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. (1989)).Phosphate-buffered saline (PBS) was prepared as described (Sambrook etal., supra). Induction medium was the same as Terrific Broth except thatit contained no salts. Agarose top was prepared by combining thefollowing reagents in a total volume of 1 liter: 10 g Bacto-tryptone, 5g yeast extract, 10 g NaCl, 1 g MgCl2.6H2O, and 7 g agarose. The mixturewas autoclaved and stored solid at room temperature. Theoligonucleotides were synthesized using the Applied Biosystems 394DNA/RNA synthesizer. DNA sequencing was performed by the dideoxy method(Sanger et al., Biotechnology, 104-108 (1992)) using the AmpliTaq DNAPolymerase FS kit and 373A DNA Sequencer Stretch (PE Applied Biosystems,Mississauga, ON, Canada). The host bacteria used for cloning was TG1:supE hsd5 thi .(lac-proAB) F′ [traD36 proAB⁺lacI^(q) lacZM15]. All thecloning steps were performed as described (Sambrook et al., supra). Thevector fd-tet was purchased from American Type Culture Collection(Manassas, Va.) and engineered such that it contained ApaI and NotIrestriction sites immediately following the gIIIp leader sequence codons(Simon J. Foote, personal communications).

[0074] Construction of Naïve Llama sdAb Library

[0075] Total RNA was isolated from the leukocytes of freshly-drawnheparinized blood of a male Llama (Lama glama) using QIAamp RNA BloodMiniTM kit (QIAGEN, Mississauga, ON, Canada) and following therecommended protocol. The concentration of RNA was calculated bymeasuring the A260 value and assuming 1 A260=40 μg/ml. Reversetranscription-polymerase chain reaction (RT-PCR) was performed on atotal of 5.3 μg RNA using the HotStarTaq PolymeraseTM kit (QIAGEN). Theprimers used included a CH2-specific primer, LlamaFOR,5′(CGCCATCAAGGTACCAGTTGA)3′ [SEQ ID No: 207] and LlamaBACK primer,5′(GATGTGCAGCTGCAGGCGTCTGGRGGAGG)3′ [SEQ ID No: 208], which anneals tothe 5′ flanking region of VH genes. Amplified product of approximately600 base pair was purified from the agarose gel using QIAquick GelExtractionTM kit (QIAGEN) and subjected to a second round of PCR usingthe primers LlamaApaII, 5′(CATGACCACAGTGCACAGGAKGTSCAGCT)3′ [SEQ ID No:209] and LlamaNotI, 5′(CGATTCTGCGGCCGCTGAGGAGACGGTGACCTG)3′ [SEQ ID No:210]. The PCR mixture contained 10 pmol/μl each of the two primers, 1×buffer (Perkin Elmer), 200 μM each of the four dNTPs and 0.05 unit/μlAmpliTaqTM DNA polymerase (Perkin Elmer). PCR protocol consisted of aninitial denaturation step at 95° C. for 15 min followed by 35 cycles of94° C. for 30 sec, 45° C. for 30 sec, and 72° C. for 1 min, and a finalextension step at 72° C. for 10 min. The primers were complimentary tothe 5′ and 3′ ends of the amplified product and incorporated ApaII andNotI restriction sites (underlined) at the end of VH genes. Theamplified products were purified using QIAquick PCR Purification kitTM(QIAGEN), cut sequentially with ApaII and NotI restrictionendonucleases, purified again, ligated to the ApaII/NotI-treated fd-tetphage vector and desalted using the above kit. Electrocompetent TG1cells were prepared (Tung et al., Trends Genet., 128-129 (1995)) and 1.5μg of the ligated product was mixed with 40 μl of competent E. colistrain TG1 and the cells were transformed by electroporation using theBIO-RAD Gene PulserTM according to the manufacturer's instructions. Thetransformed cells were immediately transferred into 1 ml of SOC mediumand split into 3 sterile tubes containing 3 ml of 50° C. agarose top,vortexed immediately, poured onto pre-warmed 2×YT petri dishes, andincubated at 37° C. overnight. The phage particles were eluted by addingfive ml of sterile PBS to the plates gently shaked at 4° C. for 3 hr.The phage-containing PBS was collected, the plates were rinsed with anadditional 5 ml PBS and the two supernatants were combined in acentrifuge bottle. The contents were centrifuged at 6000 g for 15 min at4° C., the supernatant was decanted into a sterile centrifuge bottle andthe phage was purified as described (Harrison et al., supra). At the endof the purification, the phage pellet was dissolved in 20 ml of sterilePBS and stored in liquid nitrogen in 100 μl aliquots.

[0076] To determine the size of the library, immediately following thetransformation and after the addition of the SOC medium, a small aliquotof the electroporated cells was serially diluted in exponentiallygrowing TG1 cells. 200 μl of the diluted cells was mixed with 3 ml of50° C. agarose top and immediately poured onto 2×YT plates pre-warmed to37° C. Plates were incubated overnight at 37° C. and the number ofplaques was used to determine the size of the library.

[0077] Panning

[0078] Panning was performed using the Nunc-Immuno MaxiSorpTM 8-wellstrips (Nunc). Briefly, the wells were coated overnight by adding 150 μlof 100 μg/ml antigen in PBS. In the morning, the wells were rinsed threetimes with PBS and subsequently blocked with 400 μl PBS-2% (w/v) skimmilk (2% MPBS) at 37° C. for 2 hr. The wells were rinsed as above and1012 transducing units phage in 2% MPBS were added. The mixture wasincubated at room temperature for 1.5 hr after which the unbound phagein the supematant was removed. The wells were rinsed 10 times withPBS-0.1% (v/v) Tween 20 and then 10 times with PBS to remove thedetergent. The bound phage was eluted by adding freshly prepared 200 μl100 mM triethylamine, pipetting the content of the well up and downseveral times and incubating the mixture at room temperature for 10 min.The eluted phage was transferred to a tube containing 100 μl 1 MTris-HCl, pH 7.4 and vortexed to neutralize the triethylamine. Followingthis, 10 ml of exponentially growing TG1 culture was infected with 150μl eluted phage by incubating the mixture at 37° C. for 30 min. Serialdilutions of the infected cells were used to determine the titer of theeluted phage as described in the previous section. The remainder of theinfected cells was spun down and then resuspended in 900 μl 2×YT. Thecells were mixed in 300 μl aliquots with 3 ml agarose top and the phagepropagated on the plates overnight at 37° C. In the morning the phagewas purified, the titer was determined, and a total of 10¹¹ transducingunits phage were used for further rounds of selection.

[0079] Solution Panning

[0080] Solution panning was performed using SA-PMP (1 mg/ml) obtainedfrom Promega (Madison, Wis.). To maintain SA-PMP in solution during thepanning process, the reaction tubes were flicked frequently during theincubation period. Briefly, for each target antigen 2×100 μl SA-PMPs wasfirst dispersed by gently flicking the bottom of the tubes, and thencaptured at the side of the tube in a magnetic stand (approximately 30sec.) followed by careful removal of the supematant. SA-PMPs werere-suspended in 100 μl 1× PBS, re-captured and the supernatant wasremoved. This washing process was repeated three times. To remove anypossible streptavidin binders from the phage library the phage particleswere pre-incubated with SA-PMP in 2% MPBS for 1 hr at room temperatureand the magnetic beads were captured. To form the phage-antigen complex,10¹² t.u. phage (10¹¹ t.u. for further rounds) in the supernatant wasincubated in 2% MPBS containing 20 mg/ml BSA, 0.05% Tn20 and 1 μg/mlbiotinylated antigen in a total volume of 150 μl for 1 hr at roomtemperature. In a second tube 100 μl of the washed SA-PMP was blocked in400 μl 2% MPBS at 37° C. for 2 hr. The supematant was discarded and thephage-biotinylated antigen complex solution from the first tube wasadded to the blocked SA-PMP at room temperature for 30 min. Thesupematant was removed and the complex-bound SA-PMPs were washed twicewith 100 μl PBS and then once with 100 μl 2% MPBS containing 0.05% Tn20; this sequence of washes was repeated another three times and thenfinally SA-PMPs were washed twice with PBS. The bound phage was elutedby adding 200 μl of 100 mM freshly prepared triethylamine and standingat room temperature for 10 min. Phage elution, propagation, titering andpurification were performed as described for solid phase panning.Depending on the antigen for the final third and fourth rounds theprocedure preceding the elution step was modified as described below.Following the initial washing step, 100 μl SA-PMPs were blocked followedby removal of supernatant and subsequent incubation of SA-PMPs with 100ill of 5 μg/ml biotinylated antigens in 2% MPBS at room temperature for30 min. The antigen-bound SA-PMPs were washed 5 times with 0.5% MPBS andthen incubated with phage in 2% MPBS at room temperature for 1.5 hr in atotal volume of 100 μl. The supernatant was removed and the phage boundSA-PMPs were washed eight times with 0.5% MPBS and two times with PBSbefore proceeding with the elution step.

[0081] Phage Enzyme-Linked Immunosorbent Assay (Phage ELISA)

[0082] Individual phage-infected TG1 colonies were used to inoculate 200μl of LB in sterile 96-well plates. The cells were grown overnight at100 rpm and 37° C. In the morning, the plates were spun down in a benchtop centrifuge, and the sdAb phage-containing supematant was used forphage ELISA as described below. Briefly, Nunc-Immuno MaxiSorpTM plates(Nunc) were coated overnight at 4° C. with 150 μl of 10 μg/ml of targetantigen or control proteins in PBS. The contents were removed and theplates were tapped on a paper towel to remove any liquid remaining inthe wells. The wells were blocked by adding 300 μl of PBS-2% (w/v) skimmilk (2% MPBS) and incubating for 2 hr at 37° C. The contents of thewells were emptied as before, 100 μl of sdAb phage supematant in 2% MPBSwas added, and the wells were incubated at room temperature for 1.5 hr.For biotinylated antigen, the plates were pre-coated with 5 μg/mlstreptavidin overnight followed by blocking. The wells were then coatedwith the target antigen by incubating plates with 150 μl of 1 μg/mlbiotinylated antigen at room temperature for 30 min. The wells werewashed 5× with PBS-0.05% (v/v) Tween 20 (PBST) and then incubated withphage. For control experiments no coating with the biotinylated antigenwas performed. The contents were emptied again and the wells were washed5 times with PBST and subsequently blotted on a paper towel to removeany remaining wash buffer. 100 μl of the recommended dilution ofHRP/Anti-M13 Monoclonal Conjugate (Amersham Pharmacia Biotech, Montreal,QC, Canada) in 2% MPBS was added and the wells were incubated at roomtemperature for 1 hr. The wells were washed six times as before and thebinding of sdAb to the antigen was detected colorimetrically by adding100 μl of equal mixtures of TMB Peroxidase Substrate and H2O2 (KPL,Maryland, USA) at room temperature for several min. The reaction wasstopped by adding 100 μl of 1 M H3PO4 and the A450 was measured byDYNATECH MR5000 ELISA reader (DYNATECH).

[0083] Sub-Cloning and Expression of sdAbs

[0084] sdAb genes were amplified out of the phage vector by PCR usingthe primers, VH.Bbs, 5′(TATGAAGACACCAGGCCGATGTGCAGCTGCAGGCG)3′ [SEQ IDNo: 211], and VH.Bam, 5′(TATGGATCCTGAGGAGACGGTGACCTG)3′ [SEQ ID No: 212]which also introduced BbsI and BamHI sites at the ends of the amplifiedfragments. sdAb genes were subsequently purified, cut sequentially withBbsI and BamHI restriction endonucleases, purified again with QIAquickGel ExtractionTM kit (QIAGEN), and ligated to the BbsI/BamHI-treatedpSJF-2 vector. An aliquot of the ligated product was used to transformE. coli strain TG1. Transformants were selected on ampicillin plates andthe clones harbouring the sdAb genes were identified by PCR andsequencing. For expression, single positive clones were used toinoculate 25 ml of LB containing 100 μg/ml ampicillin and the culturewas shaken at 240 rpm at 37° C. overnight. In the morning, the entireovernight culture was used to inoculate 1 liter of M9 mediumsupplemented with 5 μg/ml vitamin B1, 0.4% casamino acid and 100 μg/mlampicillin. The culture was shaken at room temperature for 30 hr at 180rpm and subsequently supplemented with 100 ml of 10× induction mediumand 100 μl of 1 M isopropylthio-D-galactoside. The culture was shakenfor another 60 hr, the periplasmic fraction was extracted by osmoticshock (Anand et al., Gene, 39-44 (1991) and the presence of sdAb in theextract was detected by Western blotting (MacKenzie et al.,Biotechnology N.Y., 390-395 (1994)). The periplasmic fraction wasdialyzed extensively in 10 mM HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) buffer pH 7.0,500 mM NaCl. The presence of the sdAb C-terminal His5 tag allowed a onestep protein purification by immobilized metal affinity chromatographyusing HiTrap ChelatingTM column (Phamacia). The 5-ml column was chargedwith Ni²⁺by applying 30 ml of a 5 mg/ml NiCl2.6H2O solution andsubsequently washed with 15 ml deionized water. Purification was carriedout as described (MacKenzie, supra) except that the starting buffer was10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the boundprotein was eluted with a 10-500 mM imidazole gradient. The purity ofthe protein was determined by SDS-PAGE (Laemmeli U.K., in: Proteases andbiological control [Reich et al., ed.], Cold Spring Harbour Laboratory,pp. 661-676 (1975)). sdAb preparation was further subjected to gelfiltration chromatography using Superdex 75 column (Pharmacia) asdescribed (Deng et al., Proc. Natl. Acad. Sci. USA., 4992-4996 (1995))and the purified monomer species were used in binding studies by surfaceplasmon resonance.

[0085] Surface Plasmon Resonance Analysis

[0086] Binding studies were performed using BIACORE 1000 (Jonsson etal., BioTechniques, 620-627 (1991)) available from Biacore Inc.,Piscataway, N.J. Binding of the anti-Yst9.1 sdAbs to Yst9.1 scFv wasassessed under the same conditions except that in this case sdAb wasimmobilized (540 RU) and the flow rate was set at 20 μl/min. For PTHbinders 186 RU (PTH2) or 70 RU (control peptide) was immobilized and theflow rate was also set at 20 μl/min. Surface regeneration was achievedby washing the sensor chips with HBST buffer. In the case of p1779binder, sdAb was passed over biotinylated p1779 (520 RU) or p1780 andp1781 control peptides (420 RU) which had been captured on a CM5 sensorchip coated with streptavidin (2260 RU). Kinetic rate constants weredetermined using BIAevaluation software and fitting to 1:1 interactionmodel. Affinity constants were calculated from the kinetic rateconstants and by Scatchard analysis of equilibrium binding data asdescribed (MacKenzie et al., J. Biol. Chem, 1527-1533 (1996)).

[0087] Although various particular embodiments of the present inventionhave been described hereinbefore for the purpose of illustration, itwould be apparent to those skilled in the art that numerous variationsmay be made thereto without departing from the spirit and scope of theinvention, as defined in the appended claims.

1 212 1 10 PRT Lama glama 1 Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 510 2 10 PRT Lama glama 2 Gly Arg Thr Phe Ser Asn Tyr His Met Gly 1 5 103 10 PRT Lama glama 3 Gly Arg Ile Phe Ser Asn Ala Ala Met Gly 1 5 10 410 PRT Lama glama 4 Arg Ser Ile Phe Ser Ile Asn Thr Leu Gly 1 5 10 5 10PRT Lama glama 5 Gly Arg Ser Phe Ser Thr Tyr Arg Val Gly 1 5 10 6 10 PRTLama glama 6 Gly Asn Thr Ile Ser Gly Tyr Ala Thr Gly 1 5 10 7 10 PRTLama glama 7 Gly Gly Ser Phe Ser Asn Tyr Asn Met Gly 1 5 10 8 10 PRTLama glama 8 Gly Arg Ile Pro Arg Asn Tyr Pro Ile Gly 1 5 10 9 10 PRTLama glama 9 Gly Glu Ser Ile Ala Ser Phe Asn Leu Gly 1 5 10 10 10 PRTLama glama 10 Gly Arg Thr Phe Ser Ser Val Ser Met Gly 1 5 10 11 10 PRTLama glama 11 Gly Leu Thr Phe Gly Asp Tyr Ala Met Gly 1 5 10 12 10 PRTLama glama 12 Gly Arg Thr Phe Ser Ser Val Thr Met Gly 1 5 10 13 10 PRTLama glama 13 Gly Arg Thr Phe Ser Arg Phe Ala Met Gly 1 5 10 14 10 PRTLama glama 14 Gly Ser Ile Phe Ser Glu Ser Ala Met Gly 1 5 10 15 10 PRTLama glama 15 Gly Arg Thr Phe Ser Ser Asp Ala Met Gly 1 5 10 16 10 PRTLama glama 16 Gly Phe Thr Phe Ser Asn Phe Trp Met Gly 1 5 10 17 10 PRTLama glama 17 Gly Arg Ser Phe Asn His Tyr Ile Met Gly 1 5 10 18 10 PRTLama glama 18 Gly Leu Pro Phe Ser Thr Tyr Ser Met Gly 1 5 10 19 10 PRTLama glama 19 Gly Arg Thr Phe Ser Thr Tyr Thr Met Gly 1 5 10 20 10 PRTLama glama 20 Gly Tyr Thr Phe Ser Ser His Ala Met Gly 1 5 10 21 10 PRTLama glama 21 Gly Phe Arg Phe Ala Glu Tyr Ala Ile Gly 1 5 10 22 10 PRTLama glama 22 Gly Arg Thr Phe Ser Arg Phe Ala Met Gly 1 5 10 23 10 PRTLama glama 23 Gly Phe Thr Phe Val Asp Tyr Ser Met Thr 1 5 10 24 10 PRTLama glama 24 Gly Phe Thr Phe Ser Asn Tyr Tyr Met Tyr 1 5 10 25 10 PRTLama glama 25 Gly Gly Thr Phe Thr Asp Tyr Ala Met Gly 1 5 10 26 10 PRTLama glama 26 Gly Gly Thr Phe Thr Asp Tyr Ala Met Gly 1 5 10 27 10 PRTLama glama 27 Gly Phe Thr Phe Ser Asn Tyr Tyr Met Tyr 1 5 10 28 10 PRTLama glama 28 Gly Asn Thr Ile Ser Asp Tyr Ala Thr Gly 1 5 10 29 17 PRTLama glama 29 Gly Ile Tyr Ser Asp Ser Ser Ile Thr Ala Tyr Ala Asp SerVal Lys 1 5 10 15 Gly 30 17 PRT Lama glama 30 Ser Ile Lys Trp Ser GlyGly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 31 17 PRT Lamaglama 31 Ala Ile Arg Trp Ser Asp Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly 32 16 PRT Lama glama 32 Trp Ile Thr Ser Gly Gly Ala ThrTyr Tyr Ala Asp Ser Met Lys Gly 1 5 10 15 33 17 PRT Lama glama 33 GlyIle Asn Trp Asn Gly Val Lys Thr Arg Tyr Ser Asp Ser Met Asn 1 5 10 15Asp 34 17 PRT Lama glama 34 Ala Val Thr Trp Ser Gly Tyr Ser Val Tyr TyrAla Lys Ser Pro Lys 1 5 10 15 Gly 35 17 PRT Lama glama 35 Gly Ile GlyTrp Ser Gly Gly Arg Ile Ile Val Ala Asp Ser Val Lys 1 5 10 15 Gly 36 17PRT Lama glama 36 Gly Ile Ser Trp Thr Ser Gly Thr Thr Tyr Phe Ala AspSer Val Lys 1 5 10 15 Gly 37 17 PRT Lama glama 37 Ala Val Ser Arg ThrGly Glu Thr Thr Asp Tyr Ala Asp Ala Val Lys 1 5 10 15 Gly 38 17 PRT Lamaglama 38 Ala Ile Asn Trp Arg Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly 39 17 PRT Lama glama 39 Thr Ile Ser Arg Ile Gly Ser ThrThr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 40 17 PRT Lama glama 40Ala Met Thr Arg Asn Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 1015 Gly 41 17 PRT Lama glama 41 Ala Ile Ser Trp Ser Gly Gly Thr Thr TyrGly Ala Asp Ser Ala Lys 1 5 10 15 Gly 42 16 PRT Lama glama 42 Ala IleThr Leu Asp Gly Arg Thr Asn Tyr Ala Tyr Tyr Ala Glu Gly 1 5 10 15 43 17PRT Lama glama 43 Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala AspSer Val Lys 1 5 10 15 Gly 44 17 PRT Lama glama 44 Gln Ile Asn Thr GlyGly Asp Ile Thr Thr Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly 45 17 PRT Lamaglama 45 Ser Ile Asp Trp Asn Ser Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys1 5 10 15 Gly 46 17 PRT Lama glama 46 Val Ile Gly Gly Gly Gly Asn ThrTyr His Ala Ala Asp Ser Leu Lys 1 5 10 15 Asp 47 17 PRT Lama glama 47Ala Ile Ser Arg Asn Ser Val Gly Thr Tyr Tyr Arg Asp Ser Val Lys 1 5 1015 Gly 48 17 PRT Lama glama 48 Ala Ile Ser Ala Ser Gly Gly Asn Gln TyrTyr Lys Tyr Phe Ala Lys 1 5 10 15 Gly 49 17 PRT Lama glama 49 Tyr IleSer Thr Ser Asp Lys Thr Thr Tyr Tyr Ser Asp Phe Ala Glu 1 5 10 15 Gly 5017 PRT Lama glama 50 Ala Ile Ser Trp Ser Gly Gly Thr Ala Tyr Gly Ala AspSer Ala Lys 1 5 10 15 Gly 51 17 PRT Lama glama 51 Ala Ile Asn Trp AsnGly Arg Leu Thr Tyr Tyr Ala Glu Ser Met Lys 1 5 10 15 Gly 52 17 PRT Lamaglama 52 Met Val Asn Thr Gly Gly Gly Gly Thr Arg Tyr Ala Asp Ser Val Arg1 5 10 15 Gly 53 17 PRT Lama glama 53 Ala Ile Ile Thr Ser Gly Arg SerThr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 54 17 PRT Lama glama 54Ala Ile Asn Trp Gly Gly Tyr Ser Thr Tyr Tyr Ser Asp Ala Val Lys 1 5 1015 Gly 55 17 PRT Lama glama 55 Met Val Asn Thr Gly Gly Gly Gly Thr ArgTyr Ala Asp Ser Val Arg 1 5 10 15 Gly 56 17 PRT Lama glama 56 Ser IleGly Arg Arg Thr Gly Trp Gln Val Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly 5712 PRT Lama glama 57 Met Val Met Gly Pro Ala Ala Thr Gly Tyr Glu Tyr 1 510 58 17 PRT Lama glama 58 Gly Ser Lys Tyr Gly Gly Ser Trp Ser Arg SerGln Asp Ala Tyr Asn 1 5 10 15 Tyr 59 17 PRT Lama glama 59 Gly Ile GlyThr Phe Gly Ser Ser Trp Thr Arg Ala Asp Arg Tyr Arg 1 5 10 15 Tyr 60 6PRT Lama glama 60 Arg Val Pro Leu Asp Tyr 1 5 61 16 PRT Lama glama 61Asp Gln Arg Phe Asp Gly Asp Asp Trp Ser Pro Ser Ala Phe Thr Arg 1 5 1015 62 16 PRT Lama glama 62 Val Phe Val Arg Thr Ala Gly Val Pro Thr LeuGly Glu Tyr Asp Tyr 1 5 10 15 63 15 PRT Lama glama 63 Thr Lys Gln PhePhe Pro Leu Ser Asn Ser Val Trp Tyr Asp Tyr 1 5 10 15 64 20 PRT Lamaglama 64 Ser Glu Arg Asp Phe Tyr Thr Arg Asn Tyr Tyr Phe Thr Phe Glu Ser1 5 10 15 Leu Tyr Asp Tyr 20 65 17 PRT Lama glama 65 Asp Tyr Asn Leu GlyThr Phe Val Thr Arg Lys Asp Ser Met Tyr Asp 1 5 10 15 Phe 66 15 PRT Lamaglama 66 Arg Arg Asn Phe Phe Gly Asn Asn Ser Ala Gly Gln Tyr Ala Tyr 1 510 15 67 12 PRT Lama glama 67 Ser Arg Tyr Val Leu Lys Tyr Asp Lys AspAla Tyr 1 5 10 68 17 PRT Lama glama 68 Lys Ala Ser Met Tyr Gly Ser ThrLeu Tyr Pro Pro Thr Gly Tyr Asn 1 5 10 15 Tyr 69 9 PRT Lama glama 69 GlyArg Ala Val Ser Asp Tyr Asp Tyr 1 5 70 13 PRT Lama glama 70 Leu Arg SerArg Ala Val Met Asp Thr Ile Pro Asn Tyr 1 5 10 71 17 PRT Lama glama 71Asp Arg Arg Arg Tyr Tyr Ser Gly Ser Tyr Pro Pro Ser Glu Tyr Asp 1 5 1015 Tyr 72 14 PRT Lama glama 72 Ala Arg Ser Val Pro Leu Ser Asp Pro ArgThr Tyr Ser Ser 1 5 10 73 14 PRT Lama glama 73 Ala Ala Ala Ala Ser ThrLeu Val Gly Gly Ser Tyr Asp Tyr 1 5 10 74 22 PRT Lama glama 74 Asp ArgAsp Phe Thr Ile Val Ala Gly Phe Ile Arg Ser Gln Tyr Ser 1 5 10 15 ProArg Ala Val Glu Tyr 20 75 15 PRT Lama glama 75 Asp Pro Met Tyr Gly ArgSer Val Met Ser Thr Arg Tyr Asn Tyr 1 5 10 15 76 18 PRT Lama glama 76Ala Thr Lys Gln Phe Ser Asn Ala Tyr Ser Asp Tyr Val His Asp Tyr 1 5 1015 Asp Tyr 77 17 PRT Lama glama 77 Gly Leu Tyr Tyr Ser Asp Tyr Arg ThrPro Glu Tyr Thr Glu Tyr Val 1 5 10 15 His 78 9 PRT Lama glama 78 Gly ArgAla Val Ser Asp Tyr Asp Tyr 1 5 79 12 PRT Lama glama 79 Gly Glu Leu TyrGly Met Gly Ser Lys His Asp Tyr 1 5 10 80 11 PRT Lama glama 80 Asp ArgPro Gln Ser Gly Trp Ser Met Asp Tyr 1 5 10 81 13 PRT Lama glama 81 ThrLys Trp Val Val Arg Arg Pro Ala Asp Tyr Asn Tyr 1 5 10 82 12 PRT Lamaglama 82 Asp Pro Gln Leu Ile Thr Thr Pro Glu Tyr Asn Tyr 1 5 10 83 11PRT Lama glama 83 Asp Arg Pro Gln Ser Gly Trp Ser Met Asp Tyr 1 5 10 8418 PRT Lama glama 84 Ser Gln Asp Ser Gly Phe Asp Thr Pro Val Thr Glu SerHis Leu Tyr 1 5 10 15 Gly Tyr 85 10 PRT Lama glama 85 Gly Phe Thr PheSer Ser Tyr Ala Met Ser 1 5 10 86 10 PRT Lama glama 86 Gly Phe Thr PheSer Ser Tyr Tyr Met Ser 1 5 10 87 10 PRT Lama glama 87 Gly Phe Thr PheAsp Glu His Ala Ile Gly 1 5 10 88 10 PRT Lama glama 88 Gly Phe Thr ValSer Ser Asn His Met Thr 1 5 10 89 10 PRT Lama glama 89 Gly Phe Thr PheSer Ser Tyr His Met Ala 1 5 10 90 10 PRT Lama glama 90 Gly Phe Thr PheAsp Glu His Ala Ile Gly 1 5 10 91 10 PRT Lama glama 91 Gly Phe Thr PheSer Arg His Gln Met Ser 1 5 10 92 10 PRT Lama glama 92 Gly Phe Thr PheArg Thr Tyr Tyr Met Asn 1 5 10 93 10 PRT Lama glama 93 Gly Phe Ile PheSer Ser Tyr Ala Met Ser 1 5 10 94 10 PRT Lama glama 94 Gly Phe Ile PheSer Ser Tyr Ala Met Ser 1 5 10 95 10 PRT Lama glama 95 Gly Phe Thr PheSer Thr Tyr Ala Met Thr 1 5 10 96 10 PRT Lama glama 96 Gly Phe Thr PheSer Ser Tyr Ala Met Ser 1 5 10 97 10 PRT Lama glama 97 Gly Phe Thr PheSer Arg His Gln Met Ser 1 5 10 98 10 PRT Lama glama 98 Gly Phe Thr PheSer Arg Tyr Ala Met Ser 1 5 10 99 10 PRT Lama glama 99 Gly Phe Thr PheSer Gly Tyr Ala Met Ser 1 5 10 100 10 PRT Lama glama 100 Gly Phe Ala PheSer Asn Tyr Arg Met Thr 1 5 10 101 10 PRT Lama glama 101 Gly Phe Thr PheSer Arg Tyr Ala Met Ser 1 5 10 102 17 PRT Lama glama 102 Gly Ile Glu GlyGly Gly Gly Ile Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 103 17 PRTLama glama 103 Thr Ile Lys Pro Gly Gly Gly Ser Thr Tyr Tyr Ala Asp SerVal Lys 1 5 10 15 Gly 104 16 PRT Lama glama 104 Thr Ile Asp Ile Gly GlyGly Arg Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 105 17 PRT Lama glama105 Arg Ile Ser Ser Asp Gly Arg Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 510 15 Gly 106 17 PRT Lama glama 106 Thr Ile Asn Pro Gly Asp Gly Ser ThrTyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 107 16 PRT Lama glama 107 HisIle Asp Thr Gly Gly Ser Thr Trp Tyr Ala Ala Ser Val Lys Gly 1 5 10 15108 16 PRT Lama glama 108 His Ile Asp Thr Gly Gly Ser Thr Trp Tyr AlaAla Ser Val Lys Gly 1 5 10 15 109 17 PRT Lama glama 109 Thr Ile Asn IleAsp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly 110 17 PRTLama glama 110 Gly Ile Asn Ser Phe Gly Gly Ser Lys Tyr Tyr Ala Asp SerVal Lys 1 5 10 15 Gly 111 17 PRT Lama glama 111 Gly Ile Asn Ser Phe GlyGly Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 112 17 PRT Lamaglama 112 Thr Ile Asn Thr Ser Gly Arg Gly Thr Tyr Tyr Ala Asp Ser ValLys 1 5 10 15 Gly 113 17 PRT Lama glama 113 Ala Ile Asn Ser Gly Gly GlySer Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 114 17 PRT Lama glama114 His Ile Asp Thr Gly Gly Gly Ser Thr Trp Tyr Ala Ala Ser Val Lys 1 510 15 Gly 115 17 PRT Lama glama 115 Asp Ile Asn Ser Gly Gly Asp Ser ThrArg Asn Ala Asp Ser Val Lys 1 5 10 15 Gly 116 17 PRT Lama glama 116 SerIle Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15Gly 117 17 PRT Lama glama 117 Arg Ile Asn Ser Ile Gly Asp Arg Ile SerTyr Ala Asp Ser Val Lys 1 5 10 15 Gly 118 17 PRT Lama glama 118 Asp IleAsn Ser Gly Gly Asp Ser Thr Arg Asn Ala Asp Ser Val Lys 1 5 10 15 Gly119 10 PRT Lama glama 119 Ala His Gly Gly Tyr Gly Ala Phe Gly Ser 1 5 10120 10 PRT Lama glama 120 Ala His Gly Gly Tyr Gly Ala Phe Gly Ser 1 5 10121 10 PRT Lama glama 121 Ala His Gly Gly Tyr Gly Ala Phe Gly Ser 1 5 10122 8 PRT Lama glama 122 Tyr Ser Gly Gly Ala Leu Asp Ala 1 5 123 8 PRTLama glama 123 Tyr Ser Gly Gly Ala Leu Asp Ala 1 5 124 8 PRT Lama glama124 Leu Ser Gln Gly Ala Met Asp Tyr 1 5 125 8 PRT Lama glama 125 Leu SerGln Gly Ala Met Asp Tyr 1 5 126 8 PRT Lama glama 126 Leu Ser Gln Gly AlaMet Asp Tyr 1 5 127 9 PRT Lama glama 127 Ile Asp Arg Glu Arg Ala Phe ThrSer 1 5 128 9 PRT Lama glama 128 Ile Asp Trp Glu Arg Ala Phe Thr Ser 1 5129 9 PRT Lama glama 129 Gln Gly Tyr Ala Gly Ser Tyr Asp Tyr 1 5 130 9PRT Lama glama 130 Leu Gly Val Pro Gly Thr Phe Asp Tyr 1 5 131 8 PRTLama glama 131 Thr Asn Arg Gly Ile Phe Asp Tyr 1 5 132 10 PRT Lama glama132 Thr Pro Gly Ser Ser Gly Val Tyr Glu Tyr 1 5 10 133 8 PRT Lama glama133 Thr Gln Thr Gly Ser His Asp Tyr 1 5 134 8 PRT Lama glama 134 Gln ValGly Thr Ala Tyr Asp Tyr 1 5 135 10 PRT Lama glama 135 Arg Arg Gly SerSer Gly Val Tyr Glu Tyr 1 5 10 136 10 PRT Lama glama 136 Gly Ser Arg ArgSer Phe Asn Val Met Gly 1 5 10 137 10 PRT Lama glama 137 Gly Asp Thr PheSer Ile Asn Ala Tyr Gly 1 5 10 138 10 PRT Lama glama 138 Gly Phe Thr PheArg Asp Tyr Trp Met Tyr 1 5 10 139 10 PRT Lama glama 139 Gly Ile Thr PheSer Glu Lys His Met Ala 1 5 10 140 10 PRT Lama glama 140 Gly Arg Thr PheSer Ser Tyr Gly Met Gly 1 5 10 141 10 PRT Lama glama 141 Gly Arg Thr PheSer Ser Tyr Gly Met Gly 1 5 10 142 10 PRT Lama glama 142 Gly Thr Ser SerGly Ile Asn Ala Met Val 1 5 10 143 10 PRT Lama glama 143 Gly Arg Thr PheSer Ser Tyr Ser Met Ala 1 5 10 144 10 PRT Lama glama 144 Val Ser Thr PheSer Ile Gly Ala Ile Gly 1 5 10 145 10 PRT Lama glama 145 Gly Ser Thr PheSer Gly Asn Asp Ile Gly 1 5 10 146 10 PRT Lama glama 146 Gly Arg Thr PheSer Ser Tyr Gly Met Gly 1 5 10 147 10 PRT Lama glama 147 Gly Arg Thr PheSer Asp Ile Ala Met Ala 1 5 10 148 10 PRT Lama glama 148 Gly Gln Thr LeuAsn Thr Tyr Val Met Gly 1 5 10 149 10 PRT Lama glama 149 Gly Pro Thr SerIle Thr Tyr Gly Met Ala 1 5 10 150 10 PRT Lama glama 150 Gly Gly Asp ValSer Thr Tyr Ala Met Val 1 5 10 151 10 PRT Lama glama 151 Gly Arg Thr PheGly Ser Tyr Thr Met Gly 1 5 10 152 10 PRT Lama glama 152 Gly Arg Thr PheSer Ser Tyr Gly Met Gly 1 5 10 153 10 PRT Lama glama 153 Gly Ser Leu SerArg Ile Thr Val Met Gly 1 5 10 154 10 PRT Lama glama 154 Gly Ser Ile SerSer Phe Asp Ala Met Ala 1 5 10 155 10 PRT Lama glama 155 Gly Arg Pro PheSer Ser Phe Ala Met Gly 1 5 10 156 10 PRT Lama glama 156 Gly Arg Thr PheSer Ser Tyr His Met Gly 1 5 10 157 16 PRT Lama glama 157 Thr Ile Thr ValGly Asp Thr Thr Ser Tyr Ala Glu Ala Val Lys Gly 1 5 10 15 158 17 PRTLama glama 158 Ala Ile Ser Gly Arg Gly Thr Asn Thr Phe Val Ala Asp SerVal Lys 1 5 10 15 Gly 159 17 PRT Lama glama 159 Ser Ile Tyr Ser Asp GlySer Arg Thr Ala Tyr Ala Ala Ser Val Lys 1 5 10 15 Gly 160 16 PRT Lamaglama 160 Val Ile Thr Arg Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val LysGly 1 5 10 15 161 16 PRT Lama glama 161 Gly Ile Thr Lys Asn Gly Val ThrTyr Tyr Ala Pro Ser Val Thr Gly 1 5 10 15 162 17 PRT Lama glama 162 AlaMet Arg Glu Ser Gly Ala Asp Thr His Tyr Ala Asp Phe Val Arg 1 5 10 15Gly 163 17 PRT Lama glama 163 Ala Met Arg Glu Ser Gly Ala Asp Thr HisTyr Ala Asp Phe Val Arg 1 5 10 15 Gly 164 16 PRT Lama glama 164 Thr IleThr Asn Ser Gly Lys Thr Asp Tyr Ala Ala Ser Ala Lys Gly 1 5 10 15 165 17PRT Lama glama 165 Ala Ile Asn Trp Arg Ser Ser Val Thr Ala Tyr Ala AspSer Val Lys 1 5 10 15 Gly 166 16 PRT Lama glama 166 Gly Ile Ser Gly GlyGly Ser Thr Tyr Tyr Thr Asp Ser Val Lys Gly 1 5 10 15 167 16 PRT Lamaglama 167 Val Ile Ser Asp Gly Gly Tyr Thr Ser Tyr Ala Thr Ser Val LysGly 1 5 10 15 168 17 PRT Lama glama 168 Ala Ile Ser Trp Gly Ala Gly ThrPro Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 169 17 PRT Lama glama 169Ala Ile Asp Trp Asn Gly Gly Thr Thr Tyr Tyr Thr Thr Phe Val Lys 1 5 1015 Gly 170 17 PRT Lama glama 170 Ala Ile Asn Trp Arg Asp Thr Ser Thr TyrTyr Gln Asp Ser Val Lys 1 5 10 15 Gly 171 17 PRT Lama glama 171 Ala ValThr Pro Ser Gly Gly Ala Ala Ala Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly172 17 PRT Lama glama 172 Leu Leu Ser Arg Ser Gly Arg Thr Thr Asn TyrAla Asp Ser Val Lys 1 5 10 15 Gly 173 16 PRT Lama glama 173 Arg Ile AsnSer Ala Gly Arg Thr Met Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 174 17 PRTLama glama 174 Ser Ile Asn Trp Arg Gly Ser Ser Thr Tyr Tyr Ala Asp SerVal Lys 1 5 10 15 Gly 175 16 PRT Lama glama 175 Ile Ile Thr Ser Ser GlyGly Thr Asp Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 176 16 PRT Lama glama176 Ile Ile Thr Ser Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 510 15 177 17 PRT Lama glama 177 Ala Ile Ser Ala Ser Gly Gly Glu Thr TyrTyr Thr Gly Ser Leu Lys 1 5 10 15 Gly 178 15 DNA Lama glamamodified_base (2) a, t, c, g, other or unknown 178 anwsgdttyy asvkg 15179 17 PRT Lama glama 179 Ala Ile Asn Trp Ser Gly Asp Thr Thr Tyr TyrGlu Ala Ser Val Lys 1 5 10 15 Gly 180 11 PRT Lama glama 180 Glu Glu TrpLeu Gly Val Arg Gln Asn Asn Tyr 1 5 10 181 3 PRT Lama glama 181 Gly GluTyr 1 182 12 PRT Lama glama 182 Met Leu Leu Gly Pro Gly Ala Pro Gly TyrAsp Tyr 1 5 10 183 9 PRT Lama glama 183 Asp Phe Tyr Gly Leu Gly Phe AspTyr 1 5 184 16 PRT Lama glama 184 Ala Pro Lys Tyr Glu Gly Val Ser AspThr Ser Ser Asp Tyr Asn Tyr 1 5 10 15 185 9 PRT Lama glama 185 Leu AspIle Thr Thr Ala Ala Ser Tyr 1 5 186 7 PRT Lama glama 186 Thr Ile Asn GlyAla Ala Arg 1 5 187 7 PRT Lama glama 187 Thr Ile Asn Gly Ala Ala Arg 1 5188 11 PRT Lama glama 188 Glu Ala Leu Pro Gly Thr Tyr Gly Leu Asp Tyr 15 10 189 9 PRT Lama glama 189 Ile Leu Ala Gly Gly Leu Leu Ala Phe 1 5190 7 PRT Lama glama 190 Gly Gly Ser Ser Gly Thr Phe 1 5 191 7 PRT Lamaglama 191 Thr Ile Asn Gly Ala Ala Arg 1 5 192 9 PRT Lama glama 192 LeuAsp Ile Thr Thr Ala Ala Ser Tyr 1 5 193 7 PRT Lama glama 193 Thr Ile AsnGly Ala Ala Arg 1 5 194 12 PRT Lama glama 194 Gly Thr Glu Leu Ala ProLys Thr Ala Thr Gly Ala 1 5 10 195 3 PRT Lama glama 195 Gly Ser Asn 1196 13 PRT Lama glama 196 Gly Thr Val Leu Ser Val Ala Thr Gly Pro TyrGly Tyr 1 5 10 197 9 PRT Lama glama 197 Trp Gly Ala Gly Glu Asp Glu AspTyr 1 5 198 12 PRT Lama glama 198 Lys Ser Arg Asp Ser Ala Gly Leu SerTrp Asp Tyr 1 5 10 199 11 PRT Lama glama 199 Leu Val Ala Ser Thr Val ThrSer Ser Val Ser 1 5 10 200 7 PRT Lama glama 200 Thr Ile Asn Gly Ala AlaArg 1 5 201 18 PRT Lama glama 201 Gln Thr Arg Pro Arg Pro Tyr Gly ThrSer Arg Ala Glu Gly Asp Tyr 1 5 10 15 Gly Tyr 202 30 PRT Lama glama 202Asp Val Lys Cys Asp Met Glu Val Ser Cys Pro Asp Gly Tyr Thr Cys 1 5 1015 Ser Arg Leu Gln Ser Gly Ala Trp Gly Cys Ser Pro Phe Thr 20 25 30 20330 PRT Lama glama 203 Val Val His Cys Asp Met Glu Val Ile Cys Pro AspGly Tyr Thr Cys 1 5 10 15 Ser Arg Leu Pro Ser Gly Ala Trp Gly Cys SerPro Phe Thr 20 25 30 204 30 PRT Lama glama 204 Asp Val Lys Cys Asp MetGlu Val Ser Cys Pro Asp Gly Tyr Thr Cys 1 5 10 15 Ser Arg Leu Pro SerGly Ala Trp Gly Cys Ser Pro Phe Thr 20 25 30 205 15 PRT Lama glama 205Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln Asp Val 1 5 10 15206 31 PRT Lama glama 206 Ser Val Ser Glu Ile Gln Leu Met His Asn LeuGly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg LysLeu Leu Gln Asp Val 20 25 30 207 21 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 207 cgccatcaag gtaccagttg a 21 208 29 DNAArtificial Sequence Description of Artificial Sequence Primer 208gatgtgcagc tgcaggcgtc tggrggagg 29 209 29 DNA Artificial SequenceDescription of Artificial Sequence Primer 209 catgaccaca gtgcacaggakgtscagct 29 210 33 DNA Artificial Sequence Description of ArtificialSequence Primer 210 cgattctgcg gccgctgagg agacggtgac ctg 33 211 35 DNAArtificial Sequence Description of Artificial Sequence Primer 211tatgaagaca ccaggccgat gtgcagctgc aggcg 35 212 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 212 tatggatcct gaggagacggtgacctg 27

What is claimed is:
 1. A phage display library of antigen-bindingfragments derived from llama antibodies, each antigen-binding fragmentcomprising at least a part of the variable heavy domain (V_(H)H or VH)of a llama antibody.
 2. A phage display library according to claim 1,wherein the antigen-binding fragment comprises a complete variable heavydomain (V_(H)H or VH).
 3. A phage display library according to claim 2,wherein the antigen-binding fragment consist essentially of a variableheavy domain (V_(H)H or VH) of a llama antibody.
 4. A phage displaylibrary according to claim 3, wherein the library is derived from theantibody repertoire of a non-immunized llama.
 5. A phage display libraryaccording to claim 4, wherein the library is of a size of at least 10⁹.6. A phage display library according to claim 5, wherein the library isof a size of at least 10⁸.
 7. A phage display library according to claim4, wherein the phage vector is a modified fd-tet phage.
 8. A phagedisplay library according to claim 7, wherein the library is generatedin the absence of a tetracycline.
 9. A phage display library accordingto claim 8, wherein the library is generated as plaques.
 10. Anantigen-binding fragment derived from a llama antibody, said fragmentcomprising at least a part of the variable heavy domain (V_(H)H or VH)of the antibody.
 11. An antigen-binding fragment according to claim 10,wherein said fragment comprises a complete variable heavy domain (V_(H)Hor VH) of the antibody.
 12. An antigen-binding fragment according toclaim 11, wherein said fragment consists essentially of the variableheavy domain (V_(H)H or VH) of a llama antibody.
 13. An antigen-bindingfragment according to claim 12, wherein the antibody is selected fromthe antibody repertoire of a non-immunized lama.
 14. An antigen-bindingfragment according to claim 13, wherein the complementarity determiningregions CDR1/H1, CDR2 and CDR3 of the variable heavy domain (V_(H)H orVH) are essentially free of cysteine residues.
 15. An antigen-bindingfragment according to claim 14, wherein the CDR1/H1 region of thevariable heavy domain (V_(H)H or VH) is selected from the groupconsisting of: GFTFSSYAMS (SEQ ID NO: 85) GFTFSSYYMS (SEQ ID NO: 86)GFTFDEHAIG (SEQ ID NO: 87) GFTVSSNHMT (SEQ ID NO: 88) GFTFSSYHMA (SEQ IDNO: 89) GFTFSRHQMS (SEQ ID NO: 91) GFTFRTYYMN (SEQ ID NO: 92) GFIFSSYAMS(SEQ ID NO: 93) GFTFSTYAMT (SEQ ID NO: 95) GFTFSGYAMS (SEQ ID NO: 99)GFAFSNYRMT (SEQ ID NO: 100) GFTFSRYAMS (SEQ ID NO: 101)


16. An antigen-binding fragment according to claim 14, wherein the CDR2region of the variable heavy domain (V_(H)H or VH) is selected from thegroup consisting of: GIEGGGGITRYADSVKG (SEQ ID NO: 102)TIKPGGGSTYYADSVKG (SEQ ID NO: 103) TIDIGGGRTYADSVKG (SEQ ID NO: 104)RISSDGRNTYYADSVKG (SEQ ID NO: 105) TINPGDGSTYYADSVKG (SEQ ID NO: 106)HIDTGGSTWYAASVKG (SEQ ID NO: 107) TINIDGSSTYYADSVRG (SEQ ID NO: 109)GINSFGGSKYYADSVKG (SEQ ID NO: 110) TINTSGRGTYYADSVKG (SEQ ID NO: 112)AINSGGGSTSYADSVKG (SEQ ID NO: 113) HIDTGGGSTWYAASVKG (SEQ ID NO: 114)DINSGGDSTRNADSVKG (SEQ ID NO: 115) SINSGGGSTYYADSVKG (SEQ ID NO: 116)RINSIGDRISYADSVKG (SEQ ID NO: 117)


17. An antigen-binding fragment according to claim 14, wherein the CDR3region of the variable heavy domain (V_(H)H or VH) is selected from thegroup consisting of: AHGGYGAFGS (SEQ ID NO: 119) YSGGALDA (SEQ ID NO:122) LSQGAMDY (SEQ ID NO: 124) IDRERAFTS (SEQ ID NO: 127) IDWERAFTS (SEQID NO: 128) QGYAGSYDY (SEQ ID NO: 129) LGVPGTFDY (SEQ ID NO: 130)TNRGIFDY (SEQ ID NO: 131) TPGSSGVYEY (SEQ ID NO: 132) TQTGSHDY (SEQ IDNO: 133) QVGTAYDY (SEQ ID NO: 134) RRGSSGVYEY (SEQ ID NO: 135)


18. An antigen-binding fragment according to claim 14, wherein saidfragment has at position 45 a residue of an amino acid other thancysteine.
 19. An antigen-binding fragment according to claim 18, whereinamino acid residues of the VL interface of the variable heavy domain(V_(H)H or VH) are Gly at position 44, Leu, Phe, Pro, or Arg at position45, and Trp, Tyr, or Phe at position
 47. 20. An antigen-binding fragmentaccording to claim 19, wherein amino acid residues at positions 44, 45and 47 are Gly, Leu and Trp, respectively.
 21. An antigen-bindingfragment according to claim 19, wherein amino acid residues at positions44, 45 and 47 are Gly, Pro and Trp, respectively.
 22. An antigen-bindingfragment according to claim 18, wherein amino acid residues of the VLinterface of the variable heavy domain (V_(H)H or VH) are Glu atposition 44, Arg at position 45, and Phe, Ile, Val, or Gly at position47.
 23. An antigen-binding fragment according to claim 18, wherein aminoacid residues of the VL interface of the variable heavy domain (V_(H)Hor VH) are Gln, Gly, Lys, Ala, or Asp at position 44, Arg at position45, and Leu, Phe, or Trp at position
 47. 24. An antigen-binding fragmentaccording to claim 18, wherein amino acid residues at positions 6, 23,74, 82a, 83, 84, 93 and 108 are Ala, Ala, Ala, Asn, Lys, Pro, Ala andGln, respectively.
 25. A cDNA library comprising nucleotide sequencescoding for antigen-binding fragments of llama antibodies, said libraryobtained by performing the steps of: (a) isolating lymphocytes from abiological sample obtained from a non-immunized llama; (b) isolatingtotal RNA from the lymphocytes; (c) reverse-transcribing and amplifyingRNA sequences coding for the antigen-binding fragments; (d) cloning theamplified cDNA in a vector, and (e) recovering the obtained clones. 26.A cDNA library according to claim 25, wherein each antigen-bindingfragment comprises at least a part of the variable heavy domain (V_(H)Hor VH) of the antibody.
 27. A cDNA library according to claim 26,wherein the antigen-binding fragment comprises a complete variable heavydomain (V_(H)H or VH) of the antibody.
 28. A cDNA library according toclaim 27, wherein the antigen-binding fragment consists essentially ofthe variable heavy domain (V_(H)H or VH) of a llama heavy chainantibody.
 29. A cDNA library according to claim 28, wherein the vectoris a filamentous bacteriophage.
 30. A cDNA library according to claim29, wherein the filamentous bacteriophage is fd-tet phage.
 31. A processfor the preparation of an antigen-binding fragment of a llama antibody,said fragment binding to a predetermined antigen, said processcomprising the steps of: (a) isolating lymphocytes from a biologicalsample obtained from a non-immunized llama; (b) isolating total RNA fromthe lymphocytes; (c) reverse-transcribing and amplifying RNA sequencescoding for antigen-binding fragments; (d) cloning the cDNA sequences soobtained into a cloning vector, said first vector capable of a surfacedisplay of the corresponding antigen-binding fragments; (e) subjectingthe clones to antigen affinity selection and recovering clones havingthe desired affinity; (f) for the recovered clones, amplifying DNAsequences coding for antigen-binding fragments; (g) cloning theamplified DNA sequences into an expression vector; (h) transforming hostcells with the expression vector under conditions allowing expression ofDNA coding for antigen binding fragments; and (i) recovering theantibody fragments having the desired specificity.
 32. A processaccording to claim 31, wherein the antigen-binding fragment comprises atleast a part of the variable heavy domain (V_(H)H or VH) of the llamaantibody.
 33. A process according to claim 32, wherein theantigen-binding fragment comprises a complete variable heavy domain(V_(H)H or VH) of the llama antibody.
 34. A process according to claim33, wherein the antigen-binding fragment consists essentially of thevariable heavy domain (V_(H)H or VH) of a llama antibody.
 35. A processaccording to claim 34, wherein the cloning vector is selected from thegroup consisting of bacteriophages, bacteria, and yeasts.
 36. A processaccording to claim 35, wherein the cloning vector is a filamentousbacteriophage.
 37. A process according to claim 36, wherein thefilamentous bacteriophage is fd-tet phage.
 38. A process according toclaim 31, wherein the expression vector is a plasmid, a phage, a virus,a YAC, or a cosmid.
 39. A process according to claim 31, wherein thehost cells are prokaryotic cells or eukaryotic cells.
 40. A processaccording to claim 39, wherein the eukaryotic cells are selected fromthe group consisting of yeast cells, mammalian cells, plant cells andprotozoan cells.